Response to Cadmium in Silene vulgaris Ecotypes Is Distinctly Affected by Priming-Induced Changes in Oxidation Status of Macromolecules.

This study investigated the impact of several priming agents on metal-tolerant and sensitive Silene vulgaris ecotypes exposed to environmentally relevant cadmium dose. We analyzed how priming-induced changes in the level of lipid, protein, and DNA oxidation contribute to calamine (Cal) and non-calamine (N-Cal) ecotype response to Cd toxicity, and whether the oxidative modifications interrelate with Cd tolerance. In non-primed ecotypes, the levels of DNA and protein oxidation were similar whereas Cal Cd tolerance was manifested in reduced lipid peroxidation. In both ecotypes protective action of salicylic acid (SA) and nitric oxide (NO) priming was observed. SA stimulated growth and reduced lipid and DNA oxidation at most, while NO protected DNA from fragmentation. Priming with hydrogen peroxide reduced biomass and induced DNA oxidation. In N-Cal, priming diminished Cd accumulation and oxidative activity, whereas in Cal, it merely affected Cd uptake and induced protein carbonylation. The study showed that priming did not stimulate extra stress resistance in the tolerant ecotype but induced metabolic remodeling. In turn, the lack of adaptive tolerance made the sensitive ecotype more responsive to the benefits of the primed state. These findings could facilitate priming exploitation with a view of enhancing metallophyte and non-metallophyte suitability for phytoremediation and land revegetation.

Differential Water Deficit in Leaves Is a Principal Factor Modifying Barley Response to Drought Stress.

In response to environmental stress, plants activate complex signalling, including being dependent on reactive oxygen-nitrogen-sulphur species. One of the key abiotic stresses is drought. As a result of drought, changes in the level of hydration of the plant occur, which obviously entails various metabolic alternations. The primary aim of this study was to determine the relationship between the response of barley to drought and the intensity of stress, therefore investigations were performed under various levels of water saturation deficit (WSD) in leaves at 15%, 30%, and 50%. In barley subjected to drought, most significant changes occurred under a slight dehydration level at 15%. It was observed that the gene expression of 9-cis-epoxycarotenoid dioxygenases, enzymes involved in ABA biosynthesis, increased significantly, and led to a higher concentration of ABA. This was most likely the result of an increase in the gene expression and enzyme activity of L-cysteine desulfhydrase, which is responsible for H2S synthesis. Our results suggest that the differential water deficit in leaves underlies the activation of an appropriate defence, with ABA metabolism at the centre of these processes. Furthermore, at 15% WSD, a dominant contribution of H2O2-dependent signalling was noted, but at 30% and 50% WSD, significant NO-dependent signalling occurred.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Efficient antioxidant defence systems of spring barley in response to stress induced jointly by the cyst nematode parasitism and cadmium exposure.

Aims: This research aimed to establish how Hordeum vulgare responds to abiotic and biotic stress affecting in tandem. Methods: Plants were inoculated with Heterodera filipjevi and treated with cadmium (Cd) concentration (5 µM) that can occur in the cultivated soil. To verify the hypothesis about participation of increased antioxidative defence in H. vulgare under stress, biochemical and microscopic methods were implemented. Results: The amount of superoxide anions and hydrogen peroxide was diminished in plants that were both nematode-inoculated and cadmium-treated. Superoxide anions were rendered harmless by increased activity of superoxide dismutase, and H2O2 was scavenged via Foyer-Halliwell-Asada pathway. The unique enhanced antioxidant capacity of double stressed plants was also linked with the accumulation of S-nitrosoglutathione as nitrosoglutathione reductase activity was inhibited. Furthermore, stimulated activity of arginase in these plants could promote polyamine synthesis and indirectly enhance non-enzymatic antioxidant mechanism. Results indicate that different antioxidants operating together significantly restricted oxidation of lipids and proteins, thus the integrity of cell membranes and protein functions were maintained. Conclusions: The ROS deactivation machinery in barley leaves showed an unusual response during stress induced by H. filipjevi infection and cadmium treatment. Plants could induce a multi-component model of stress response, to detoxify Cd ions and efficiently repair stress damage.

Reactive oxygen species metabolism and photosynthetic performance in leaves of Hordeum vulgare plants co-infested with Heterodera filipjevi and Aceria tosichella.

KEY MESSAGE: Defence responses of cyst nematode and/or wheat curl mite infested barley engage the altered reactive oxygen species production, antioxidant machinery, carbon dioxide assimilation and photosynthesis efficiency. The primary aim of this study was to determine how barley responds to two pests infesting separately or at once; thus barley was inoculated with Heterodera filipjevi (Madzhidov) Stelter (cereal cyst nematode; CCN) and Aceria tosichella Keifer (wheat curl mite; WCM). To verify hypothesis about the involvement of redox metabolism and photosynthesis in barley defence responses, biochemical, photosynthesis efficiency and chlorophyll a fluorescence measurements as well as transmission electron microscopy were implemented. Inoculation with WCM (apart from or with CCN) brought about a significant suppression in the efficiency of electron transport outside photosystem II reaction centres. This limitation was an effect of diminished pool of rapidly reducing plastoquinone and decreased total electron carriers. Infestation with WCM (apart from or with CCN) also significantly restricted the electron transport on the photosystem I acceptor side, therefore produced reactive oxygen species oxidized lipids in cells of WCM and double infested plants and proteins in cells of WCM-infested plants. The level of hydrogen peroxide was significantly decreased in double infested plants because of glutathione-ascorbate cycle involvement. The inhibition of nitrosoglutathione reductase promoted the accumulation of S-nitrosoglutathione increasing antioxidant capacity in cells of double infested plants. Moreover, enhanced arginase activity in WCM-infested plants could stimulate synthesis of polyamines participating in plant antioxidant response. Infestation with WCM (apart from or with CCN) significantly reduced the efficiency of carbon dioxide assimilation by barley leaves, whereas infection only with CCN expanded photosynthesis efficiency. These were accompanied with the ultrastructural changes in chloroplasts during CCN and WCM infestation.

Cyst Nematode Infection Elicits Alteration in the Level of Reactive Nitrogen Species, Protein S-Nitrosylation and Nitration, and Nitrosoglutathione Reductase in Arabidopsis thaliana Roots.

Reactive nitrogen species (RNS) are redox molecules important for plant defense against pathogens. The aim of the study was to determine whether the infection by the beet cyst nematode Heterodera schachtii disrupts RNS balance in Arabidopsis thaliana roots. For this purpose, measurements of nitric oxide (NO), peroxynitrite (ONOO-), protein S-nitrosylation and nitration, and nitrosoglutathione reductase (GSNOR) in A. thaliana roots from 1 day to 15 days post-inoculation (dpi) were performed. The cyst nematode infection caused generation of NO and ONOO- in the infected roots. These changes were accompanied by an expansion of S-nitrosylated and nitrated proteins. The enzyme activity of GSNOR was decreased at 3 and 15 dpi and increased at 7 dpi in infected roots, whereas the GSNOR1 transcript level was enhanced over the entire examination period. The protein content of GSNOR was increased in infected roots at 3 dpi and 7 dpi, but at 15 dpi, did not differ between uninfected and infected roots. The protein of GSNOR was detected in plastids, mitochondria, cytoplasm, as well as endoplasmic reticulum and cytoplasmic membranes. We postulate that RNS metabolism plays an important role in plant defense against the beet cyst nematode and helps the fine-tuning of the infected plants to stress sparked by phytoparasitic nematodes.

Effects of lead, cadmium and zinc on protein changes in Silene vulgaris shoots cultured in vitro.

In the present research, Silene vulgaris as a representative species growing on both unpolluted and heavy metal (HM) polluted terrains were used to identify ecotype-specific responses to metallic stress. Growth, cell ultrastructure and element accumulations were compared between non-metallicolous (NM), calamine (CAL) and serpentine (SER) specimens untreated with HMs and treated with Pb, Cd and Zn ions under in vitro conditions. Moreover, proteins' modifications related to their level, carbonylation and degradations via vacuolar proteases were verified and linked with potential mechanisms to cope with ions toxicity. Our experiment revealed diversified strategy of HM uptake in NM and both metallicolous ecotypes, in which antagonistic relationship of Zn and Pb/Cd ions provided survival benefits for the whole organism. Despite this similarity, growth rate and metabolic pathways induced in CAL and SER shoots varied significantly. Exposition to HMs in CAL culture led to drop in protein level by approximately 16% compared to the control. This parameter nearly correlated with the enhanced activity of proteases at pH 5.2 as well as possible glutamate changes to proline and reduced glutathione, resulting in intensified growth and first signs of cell senescence. In turn, SER shoots were characterized by growth retardation (to 53% of the control), although protein level and carbonylation were not modified, while a deeper insight into protein network showed its remodeling towards production of polyamines and 2-oxoglutarate delivered to the Krebs cycle. Contrary, an uncontrolled HM influx in NM shoots contributed to morpho-structural disorders accompanied by an increase activity of proteases involved in the degradation of oxidized proteins, what pointed to metal-induced autophagy. Taken together, S. vulgaris ecotypes respond to stress by triggering various mechanisms engaged their survival and/or death under HM treatment.

Ecotype-Specific Pathways of Reactive Oxygen Species Deactivation in Facultative Metallophyte Silene Vulgaris (Moench) Garcke Treated with Heavy Metals.

This research aimed to indicate mechanisms involved in protection against the imbalanced generation of reactive oxygen species (ROS) during heavy metals (HMs) exposition of Silene vulgaris ecotypes with different levels of metal tolerance. Specimens of non-metallicolous (NM), calamine (CAL), and serpentine (SER) ecotypes were treated in vitro with Zn, Pb, and Cd ions applied simultaneously in concentrations that reflected their contents in natural habitats of the CAL ecotype (1× HMs) and 2.5- or 5.0-times higher than the first one. Our findings confirmed the sensitivity of the NM ecotype and revealed that the SER ecotype was not fully adapted to the HM mixture, since intensified lipid peroxidation, ultrastructural alternations, and decline in photosynthetic pigments' content were ascertained under HM treatment. These changes resulted from insufficient antioxidant defense mechanisms based only on ascorbate peroxidase (APX) activity assisted (depending on HMs concentration) by glutathione-S-transferase (GST) and peroxidase activity at pH 6.8 in the NM ecotype or by GST and guaiacol-type peroxidase in the SER one. In turn, CAL specimens showed a hormetic reaction to 1× HMs, which manifested by both increased accumulation of pigments and most non-enzymatic antioxidants and enhanced activity of catalase and enzymes from the peroxidase family (with the exception of APX). Interestingly, no changes in superoxide dismutase activity were noticed in metallicolous ecotypes. To sum up, the ROS scavenging pathways in S. vulgaris relied on antioxidants specific to the respective ecotypes, however the synthesis of polyphenols was proved to be a universal reaction to HMs.

Activity profiling of barley vacuolar processing enzymes provides new insights into the plant and cyst nematode interaction.

Vacuolar processing enzymes (VPEs) play an important role during regular growth and development and defence responses. Despite substantial attempts to understand the molecular basis of plant-cyst nematode interaction, the mechanism of VPEs functioning during this interaction remains unknown. The second-stage Heterodera filipjevi juvenile penetrates host roots and induces the formation of a permanent feeding site called a syncytium. To investigate whether infection with H. filipjevi alters plant host VPEs, the studies were performed in Hordeum vulgare roots and leaves on the day of inoculation and at 7, 14 and 21 days post-inoculation (dpi). Implementing molecular, biochemical and microscopic methods we identified reasons for modulation of barley VPE activity during interaction with H. filipjevi. Heterodera filipjevi parasitism caused a general decrease of VPE activity in infected roots, but live imaging of VPEs showed that their activity is up-regulated in syncytia at 7 and 14 dpi and down-regulated at 21 dpi. These findings were accompanied by tissue-specific VPE gene expression patterns. Expression of the barley cystatin HvCPI-4 gene was stimulated in leaves but diminished in roots upon infestation. External application of cyclotides that can be produced naturally by VPEs elicits in pre-parasitic juveniles vesiculation of their body, enhanced formation of granules, induction of exploratory behaviour (stylet thrusts and head movements), production of reactive oxygen species (ROS) and final death by methuosis. Taken together, down-regulation of VPE activity through nematode effectors promotes the nematode invasion rates and leads to avoidance of the induction of the plant proteolytic response and death of the invading juveniles.

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii.

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter Rfd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.

Protease activity and phytocystatin expression in Arabidopsis thaliana upon Heterodera schachtii infection.

The activity of plant proteases is important for amino acids recycling, removal of damaged proteins as well as defence responses. The second-stage juvenile of the beet cyst nematode Heterodera schachtii penetrates host roots and induces the feeding site called a syncytium. To determine whether infection by H. schachtii affects proteolysis, the protease activity was studied in Arabidopsis roots and shoots at the day of inoculation and 3, 7 and 15 days post inoculation (dpi). Nematode infection caused a decrease of protease activities in infected roots over the entire examination period at all studied pH values. In contrast, the activities of the low molecular mass as well as Ca2+-dependent cysteine proteases were found to be stimulated. In shoots of infected plants, the protease activity was diminished only at 15 dpi at all tested pH values. It was accompanied by changes in total soluble protein content, a higher protein carbonylation and a total polyphenol content. To go deeper into proteolysis regulation, the expression of phytocystatin genes, endogenous inhibitors of cysteine proteases, was examined in syncytia, roots and shoots. Expression of AtCYS1, AtCYS5 and AtCYS6 genes was enhanced upon cyst nematode infection. Our results suggest that changes in protease activities in roots and shoots and altered cystatin expression patterns in syncytia, roots and shoots are important for protein metabolism during cyst nematode infection.

[Biochemical mechanisms of neurotoxicity caused by cadmium]. / Biochemiczne mechanizmy neurotoksycznosci kadmu.

Cadmium (Cd), which belongs to the heavy metals, is one of the major polluting component of human and animal environment. Exposure to cadmium can lead to absorption of the compounds to the organism and consequently, the toxic effects in the nervous system. The paper presents various views on the biochemical mechanisms of neurotoxicity caused by cadmium. This paper describes the disturbances in the cellular antioxidant system, generation of reactive oxygen and nitrogen species, changes in energy production in the metabolic pathways, changes in the metabolism of biogenic amines, neurotransmitter amino acids and calcium ions and inhibition of enzymatic proteins.