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

Intracelluláris stresszválaszok növényekben és azok leképezése: a nitrogén-monoxid szerepe = Intracellular stress responses in plants and their imaging: the role of nitric oxide

A nitrogén-monoxid (NO) hatékony jelátviteli molekula a növények környezeti hatásokra adott gyors válaszreakcióiban. Pályázatunkban a NO szerepét tanulmányoztuk: a gyökér architektúra fejlődésében ozmotikus stressz alatt; a fotoszintetikus elektron transzportlánc működésének szabályozásában és a nehéz fémek (NF) által előidézett stressz-válaszban. Az ozmotikus stressz- és az auxin-indukálta NO képződés különböző jellegű folyamatok a gyökér fejlődésében: az auxin-indukálta gyökér architektúra fejlődést a NO képződés párhuzamosan kísérte, ozmotikus stressz alatt ezt a folyamatot egy gyors, tranziens NO képződés előzte meg. Ez a tranziens búza esetében is megjelent. Arabidopsis nitrát reduktáz (NR) hiánymutánsok (nia1, nia2) és Atnoa1(korábban Atnos1) mutáns segítségével kimutattuk, hogy a NO forrása az auxin-indukálta gyökér fejlődés során a NR. A fotoszintetikus elektron transzportláncra vonatkozólag in vivo klorofill fluoreszcencia mérések szerint a NO gátolja a steady-state fotokémiai és nem-fotokémiai kioltási reakciókat és modulálja a reakciócentrumhoz kapcsolódó nem-fotokémiai kioltást. A különböző NF fajták, mint a Cu2+ és a Cd2+, különböző mértékben idézik elő a NO képződését. Réz ionok esetében két-fázisú kinetika jelent meg; a kezelés utáni az első 6 órában gyors NO tranziens után lassú, fokozatosan növekvő NO szint jelent meg. Ez a tranziens új jelenség, melyet még nem írtak le a növények nehézfém-stressz alatti válaszreakcióiban. | Nitrogen oxide (NO) is a potent signalling molecule in fast responses to environmental stimuli in plants. Three types of stress responses were investigated: the role of NO and its source in the development of root architecture under osmotic stress, in the regulation of photosynthetic electron transport, and in heavy metal (HM) stress responses. We found that osmotic stress- and the auxin-induced NO generations were distinct processes in root development in pea plants since changes in root architecture occurred in parallel with an intensified NO generation while under osmotic stress they were preceded by a transient burst of NO. The early NO burst appeared also in wheat. As for the source of NO, using nitrate reductase (NR)-deficient nia1, nia2 and Atnoa1(former Atnos1) mutants of Arabidopsis, we showed out that NR is responsible for exogenous auxin-induced NO synthesis. In the regulation of photosynthetic electron transport as studied by in vivo chlorophyll fluorescence detection, NO inhibited the steady-state photochemical and non-photochemical quenching processes and appeaed to modulate reaction-center-associated non-photochemical quenching. For the NO production the most effective metals were Cu2+ and Cd2+; in case of Cu2+, a fast NO burst appeared in the first six hours followed by a slower, gradual increase. The fast appearance of NO in the presence of cupric ions suggests a novel reaction hitherto not studied in plants under heavy metal stress

Development of Ash Dieback in South-Eastern Germany and the Increasing Occurrence of Secondary Pathogens

Since its first identification in Poland in 2006, the ascomycete Hymenoscyphus fraxineus has caused massive dieback of Fraxinus excelsior in the countries of eastern, northern and central Europe. This work shows the development, expansion, and severity of the disease in south-eastern Germany for a period of four years, starting in 2010. Differences between habitats, as well as age classes have been captured. The presence and the amount of potentially resistant trees were proven over the years, to determine how high the resistance level might be. Typical disease symptoms are the wilting of leaves, necrotic lesions in the bark and reddish discolorations of branches and stems. In addition, stem necroses also appear by infection with species of Armillaria. Therefore, special attention has been given to Armillaria species in affected ash stands but also to other secondary pathogens, like ash bark beetles. It is shown that breeding galleries of Hylesinus fraxini are only found in trees that have recently died and thus Hylesinus fraxini is still acting as a secondary opportunistic pathogen. In contrast, Armillaria spp. can be considered as serious pathogens of weakened ash trees. In different ash stands, typical symptoms of infection can be found. A relationship between stem base necrotic lesions and vitality was examined. It is shown that necrotic lesions severely contribute to accelerating the mortality of ash trees. In addition to the high infection pressure by H. fraxineus, the high inoculum of Armillaria in the soil facilitates further infections and, thus, likewise endangers the survival of potentially resistant trees. In the following years, forest conversion and seed harvest in affected ash stands will have to be urgently considered to avoid tree gaps on a large scale. Furthermore, infection assays of potentially resistant trees with ensuing breeding programmes should be initially started for the conservation of this ecologically and economically important tree species

Spatio-Temporal Monitoring of Benthic Anatoxin-a-Producing Tychonema sp. in the River Lech, Germany

Incidents with toxic benthic cyanobacteria blooms have been increasing recently. In 2019, several dogs were poisoned in the river Lech (Germany) by the benthic anatoxin-a-producing genus Tychonema. To characterize spatial and temporal distribution of potentially toxic Tychonema in this river, a systematic monitoring was carried out in 2020, focusing on the occurrence of the genus, its toxin production and habitat requirements. Tychonema and cyanobacterial community composition in benthic mats and pelagic samples were identified using a combined approach of microscopy and DNA sequencing of the 16S rRNA gene. In addition, anatoxin-a concentrations of selected samples were measured using the ELISA method. The habitat was characterized to assess the ecological requirements and growth conditions of Tychonema. Tychonema mats and anatoxin-a were detected at several sampling sites throughout the entire study period. Toxin concentrations increased with the progression of the vegetation period and with flow direction, reaching values between 0 and 220.5 µg/L. Community composition differed among pelagic and benthic samples, with life zone and substrate condition being the most important factors. The results of this study highlight the importance of monitoring and understanding the factors determining occurrence and toxin production of both pelagic and benthic cyanobacteria due to their relevance for the health of humans and aquatic ecosystems

Effects of cellulase-containing enzymes on auxin heterotrophic and autotrophic tobbaco tissue cultures

ABSTRACT During the digestion of cell wall by cellulases, rapid cell death of auxin heterotrophic and autotrophic tobacco tissue cells has been observed. Under these conditions a considerable amount of H 2 O 2 is secreted into the medium. It is suggested that the rapid cell death induced by cellullysin is partly a result of the hypersensitive defense reaction against cellulysin from the parasitic fungus Trichoderma viride. The absolute amount of secreted H 2 O 2 is higher in the case of heterotrophic cells, but the quantity of H 2 O 2 is greater in the medium of auxin autotrophic cultures as compared with control cells without cellulysin. These cells emit more cellulase-dependent ethylene than the auxin heterotrophic line. Cellulysin induces an increasing intracellular H 2 O 2 level only in the cells of auxin heterotrophic tissues. There are differences between the two cell lines in the change in cytosolic Ca 2+ concentration in response to exogenous H 2 O 2

Mass Occurrence of Anatoxin-a- and Dihydroanatoxin-a-Producing Tychonema sp. in Mesotrophic Reservoir Mandichosee (River Lech, Germany) as a Cause of Neurotoxicosis in Dogs

In August 2019, three dogs died after bathing in or drinking from Mandichosee, a mesotrophic reservoir of the River Lech (Germany). The dogs showed symptoms of neurotoxic poisoning and intoxication with cyanotoxins was considered. Surface blooms were not visible at the time of the incidents. Benthic Tychonema sp., a potential anatoxin-a (ATX)-producing cyanobacterium, was detected in mats growing on the banks, as biofilm on macrophytes and later as aggregations floating on the lake surface. The dogs’ pathological examinations showed lung and liver lesions. ATX and dihydroanatoxin-a (dhATX) were detected by LC-MS/MS in the stomachs of two dogs and reached concentrations of 563 and 1207 µg/L, respectively. Anatoxins (sum of ATX and dhATX, ATXs) concentrations in field samples from Mandichosee ranged from 0.1 µg/L in the open water to 68,000 µg/L in samples containing a large amount of mat material. Other (neuro)toxic substances were not found. A molecular approach was used to detect toxin genes by PCR and to reveal the cyanobacterial community composition by sequencing. Upstream of Mandichosee, random samples were taken from other Lech reservoirs, uncovering Tychonema and ATXs at several sampling sites. Similar recent findings emphasize the importance of focusing on the investigation of benthic toxic cyanobacteria and applying appropriate monitoring strategies in the future