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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, H 2 O 2 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., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The well-known neurotransmitter 5-hydroxytryptamine (serotonin) not only regulates sleep and mood in humans and animals but may also play important roles in modulating growth, development, and defense responses, such as seed germination, flowering, and abiotic stress tolerance, in plants. Serotonin inhibits primary root (PR) growth; however, the physiological and molecular mechanisms underlying serotonin-mediated PR growth inhibition remain largely unclear. Here, we investigate the effects of serotonin on root growth and development in Arabidopsis. Serotonin inhibits PR elongation by affecting both the meristem and elongation zones. In the meristem zone, serotonin represses both meristem cell division potential and stem cell niche activity. Serotonin induces H 2 O 2 overaccumulation in the elongation zone and reduces O 2 - accumulation in the meristem zone by a UPB1 pathway, thereby disrupting reactive oxygen species (ROS) equilibrium in root tips, thus resulting in PR growth inhibition. Serotonin also regulates auxin distribution in root tips by decreasing auxin-related gene expression and repressing auxin transport through modulation of AUX1 and PIN2 abundances in root tips. Taken together, our data indicate that high concentrations of serotonin result in stress responses in plants by inhibiting PR elongation through the regulation of H 2 O 2 and O 2 - distribution in PR tips and through an auxin pathway via the repression of auxin biosynthesis and transport., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Exposure of barley roots to mM concentrations of L-NAME for 30 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 H 2 O 2 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., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Enhanced superoxide generation and significant inhibition of succinate dehydrogenase (SDH) activity followed by a strong reduction of root growth were detected in barley seedlings exposed to a 5μM Hg concentration for 30min, which increased further in an Hg dose-dependent manner. While at a 25μM Hg concentration no cell death was detectable, a 50μM Hg treatment triggered cell death in the root meristematic zone, which was markedly intensified after the treatment of roots with 100μM Hg and was detectable in the whole root tips. Generation of superoxide and H 2 O 2 was a very rapid response of root tips occurring even after 5min of exposure to Hg. Application of an NADPH oxidase inhibitor or the inhibition of electron flow in mitochondria by the inhibition of complex I did not influence the Hg-induced H 2 O 2 production. Treatment of roots with thenoyltrifluoroacetone, a non-competitive inhibitor of SDH, markedly reduced root growth and induced both superoxide and H 2 O 2 production in a dose dependent manner. Similar to results obtained in intact roots, Hg strongly inhibited SDH activity in the crude mitochondrial fraction and caused a considerable increase of superoxide production, which was markedly reduced by the competitive inhibitors of SDH. These results indicate that the mitochondrial complex II-derived superoxide is the primary source of Hg toxicity in the barley root tip., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The research was conducted on embryo axes of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), which were isolated from imbibed seeds and cultured for 96h in vitro under different conditions of carbon and nitrogen nutrition. Isolated embryo axes were fed with 60mM sucrose or were sugar-starved. The effect of 35mM asparagine (a central amino acid in the metabolism of germinating lupin seeds) and 35mM nitrate (used as an inorganic kind of nitrogen) on growth, storage lipid breakdown and autophagy was investigated. The sugar-starved isolated embryo axes contained more total lipid than axes fed with sucrose, and the content of this storage compound was even higher in sugar-starved isolated embryo axes fed with asparagine. Ultrastructural observations showed that asparagine significantly slowed down decomposition of autophagic bodies, and this allowed detailed analysis of their content. We found peroxisomes inside autophagic bodies in cells of sugar-starved Andean lupin embryo axes fed with asparagine, which led us to conclude that peroxisomes may be degraded during autophagy in sugar-starved isolated lupin embryo axes. One reason for the slower degradation of autophagic bodies was the markedly lower lipolytic activity in axes fed with asparagine., (Copyright © 2016 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca 2+ -activated states., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

We aimed to reveal the mechanisms of mitotic spindle anomalies induced by microcystin-LR (MCY-LR), a cyanobacterial toxin in Vicia faba, a well-known model in plant cell and molecular biology. MCY-LR inhibits type 1 and 2A phosphoserine/threonine specific protein phosphatases (PP1 and PP2A) and induces reactive oxygen species (ROS) formation. The cytoskeleton is one of the main targets of the cyanotoxin during cytopathogenesis. Histochemical-immunohistochemical and biochemical methods were used. A significant number of MCY-LR induced spindle alterations are described for the first time. Disrupted, multipolar spindles and missing kinetochore fibers were detected both in metaphase and anaphase cells. Additional polar microtubule (MT) bundles, hyperbundling of spindle MTs, monopolar spindles, C-S- shaped, additional and asymmetric spindles were detected in metaphase, while midplane kinetochore fibers were detected in anaphase cells only. Several spindle anomalies induced mitotic disorders, i.e. they occurred concomitantly with altered sister chromatid separation. Alterations were dependent on the MCY-LR dose and exposure time. Under long-term (2 and mainly 6 days') exposure they were detected in the concentration range of 0.1-20μgmL(-1) MCY-LR that inhibited PP1 and PP2A significantly without significant ROS induction. Elevated peroxidase/catalase activities indicated that MCY-LR treated V. faba plants showed efficient defense against oxidative stress. Thus, although the elevation of ROS is known to induce cytoskeletal aberrations in general, this study shows that long-term protein phosphatase inhibition is the primary cause of MCY-LR induced spindle disorders., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Long-term treatment of Allium cepa seedlings with low concentration of hydroxyurea (HU) results in a disruption of cell cycle checkpoints, leading root apex meristem (RAM) cells to an abnormal organization of nuclear structures forming interphase (I) and mitotic (M) domains of chromatin at opposite poles of the nucleus. Thus far, both critical cell length and an uneven distribution of cyclin B-like proteins along the nuclear axis have been recognized as essential factors needed to facilitate the formation of biphasic interphase-mitotic (IM) cells. Two new aspects with respect to their emergence are investigated in this study. The first concerns a relationship between the polarity of increasing chromatin condensation (IM orientation) and the acropetal (base→apex) alignment of RAM cell files. The second problem involves the effects of auxin (IAA), on the frequency of IM cells. We provide evidence that there is an association between the advanced M-poles of the IM cell nuclei and the polarized accumulation sites of auxin efflux carriers (PIN2 proteins) and IAA. Furthermore, our observations reveal exclusion regions for PIN2 proteins in the microtubule-rich structures, such as preprophase bands (PPBs) and phragmoplast. The current and previous studies have prompted us to formulate a hypothetical mechanism linking PIN2-mediated unilateral localization of IAA and the induction of bipolar IM cells in HU-treated RAMs of A. cepa., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Auxin is a master regulator of root growth by modulating its development under the constantly changing environment. Recently, an antagonistic interaction was suggested between SA and IAA signaling. Therefore, the purpose of this work was to analyze and compare the effect of the indole-3-acetic acid (IAA) signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) and salicylic acid (SA) as a potential IAA signaling inhibitor on the root growth, enzyme activity and reactive oxygen species (ROS) production in Cd- and IAA-treated barley root tips. Exposure of plants to Cd resulted in a more than threefold increase of IAA content in the root apex even 3h after the treatment. In addition, exogenously applied IAA evoked root responses such as root growth inhibition and swelling, ROS generation and activation of lipoxygenase or glutathione peroxidase identical to those induced by Cd. Furthermore, both Cd- and IAA-induced stress responses were markedly reversed by PCIB or SA post-treatment. Similarly to PCIB, SA did not affect the IAA content of root tips, suggesting the action of SA on the IAA signaling pathway in barley roots. SA probably does not alleviate the Cd toxicity in roots, but rather prevents or partially inhibits the root defense response to the presence of Cd through the inhibition of Cd-induced IAA-mediated ROS generation in roots., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Tungsten is a heavy metal with increasing concern over its environmental impact. In plants it is extensively used to deplete nitric oxide by inhibiting nitrate reductase, but its presumed toxicity as a heavy metal has been less explored. Accordingly, its effects on Arabidopsis thaliana primary root were assessed. The effects on root growth, mitotic cell percentage, nitric oxide and hydrogen peroxide levels, the cytoskeleton, cell ultrastructure, auxin and cytokinin activity, and auxin carrier distribution were investigated. It was found that tungsten reduced root growth, particularly by inhibiting cell expansion in the elongation zone, so that root hairs emerged closer to the root tip than in the control. Although extensive vacuolation was observed, even in meristematic cells, cell organelles were almost unaffected and microtubules were not depolymerized but reoriented. Tungsten affected auxin and cytokinin activity, as visualized by the DR5-GFP and TCS-GFP expressing lines, respectively. Cytokinin fluctuations were similar to those of the mitotic cell percentage. DR5-GFP signal appeared ectopically expressed, while the signals of PIN2-GFP and PIN3-GFP were diminished even after relatively short exposures. The observed effects were not reminiscent of those of any nitric oxide scavengers. Taken together, inhibition of root growth by tungsten might rather be related to a presumed interference with the basipetal flow of auxin, specifically affecting cell expansion in the elongation zone., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Root growth inhibition and radial root swelling were the characteristic symptoms of barley root tips after the short-term exposure of roots to 15 and 30μM Cd. Higher Cd concentrations caused extensive cell death and root growth arrest. Enhanced lipid peroxidation was observed as early as 1h after the short-term treatment in a Cd concentration-dependent manner. In contrast to lipid peroxidation, the induction of lipoxygenase activity was detected only 3h after the exposure of roots to 15 or 30μM Cd. In addition, it was not observed in 60μM Cd-treated root tips. The highest lipoxygenase activity was detected 6h after 15μM Cd treatment in the meristematic and elongation zone of root tip and was probably associated with the radial expansion of cells. Our results indicate that the upregulation of lipoxygenase is an important component of stress response in barley roots to toxic Cd. It is probably involved in the morphological stress response of root tips or/and in the alleviation of Cd-induced toxic alterations in plant cell membranes, but it is not responsible for the Cd-induced harmful lipid peroxidation and cell death., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Carbohydrate starvation of Vicia faba root meristems leads to readjustment of carbohydrate metabolism and blocks the cell cycle in two principal control points (PCP1/2). The cell cycle reactivation is possible after sucrose provision, although with a delay of about 12h. During this period, the cells are sensitive to 6-dimethylaminopurine (6-DMAP) and okadaic acid (OA), inhibitors of protein kinases and phosphatases, respectively. The aim of the present study was to investigate whether those inhibitors are involved in inhibition of cell cycle revival through interference with the activities of two sucrose-cleaving enzymes: sucrose synthase (SuSy; EC 2.4.1.13) and invertase (INV; EC 3.2.1.26). In sugar-starved cells, the in situ activity of both enzymes decreased significantly. Following supplementation of root meristems with sugar, INV remained inactive, but SuSy activity increased. Despite the lack of INV activity, glucose was present in meristem cells, but its content was low in cells treated with OA. In the latter case, the size of plastids was reduced, they had less starch, and Golgi structures were affected. In sugar-starved cells, SuSy activity was induced more by exogenous sucrose than by glucose. The sucrose-induced activity was strongly inhibited by OA (less by 6-DMAP) at early stages of regeneration, but not at the stages preceding DNA replication or mitotic activities. The results indicate that prolongation of regeneration and a marked decrease in the number of cells resuming proliferation (observed in previous studies) and resulting from the action of inhibitors, are correlated with the process of SuSy activation at the beginning of regeneration from sugar starvation., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Short-term treatment (30 min) of barley roots with a low 10 μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60 μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6 h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10 μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60 μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The effect of Cd on NADPH-diaphorase activity and nitric oxide (NO) production was investigated in barley root tips. The Cd-induced increase of NADPH-diaphorase activity occurred at the elongation zone and increased further in the differentiation zone of barley root tips. This activity was associated primarily with the microsomal membrane fraction of crude extract. In situ analysis revealed that the diaphorase activity was localized in the metaxylem and metaphloem elements and to some cells of the pericycle and parenchyma of root tips. Cd-induced NO generation was observed in pericycle, parenchymatic stelar cells and companion cells of protophloem. The results suggest that the Cd-induced generation of NO functions in Cd toxicity through the ectopic and accelerated differentiation of root tips, causing the shortening of the root elongation zone and a subsequent reduction in root growth.

DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20kDa (OsCyt20) and a nuclear of 37kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca(2+)/Mg(2+)-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn(2+)-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.

Previous research has reported inconsistent results from experiments on the influence of boron (B) on plant sensitivity to potentially toxic aluminium (Al) concentrations. Differences in B requirement and cell wall properties among species, especially between Poaceae and dicots, may account for this. This investigation reports amelioration by B of Al-induced inhibition of root elongation in Al-sensitive cucumber (Cucumis sativus), but not in Al-sensitive maize (Zea mays). Vital staining, however, also revealed a positive influence of B supply on Al tolerance in maize. In both species, adequate B supply decreased Al-induced damage of cell integrity. In cucumber, increasing B supply enhanced Al concentrations and haematoxylin staining in root tips. In maize, no differences for root Al among B treatments were observed. These results indicate that the positive effect of B on Al resistance was not due to less Al accumulation in root tips. Enhanced concentrations of reduced glutathione were found in roots of Al-stressed maize plants growing with adequate B. It is concluded that adequate B supply is essential for prevention of Al toxicity in both the dicot and the monocot species. In dicot cucumber, the B-induced amelioration of root elongation, despite higher Al accumulation in root tips, indicates B-induced change in either or both Al speciation and compartmentation in the tips. The protection by an adequate B supply of roots against Al-induced cell death suggests a role for B in the defence against oxidative stress. This is supported by the observation that Al induced enhanced levels of GSH in roots of maize plants growing with adequate B supply but not in those growing with either deficient or excess B concentrations.

Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.

Responses to Al3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al3+ toxicity and resistance. A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al3+ ion in culture media, which was supplied as 0.08-2.3 mM Al3+ for 2-8 weeks. Increasing Al3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells. Exposure to 0.8 mM Al3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected. In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis. The Al-R cells appeared to exclude Al3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R). The results showed that by studying the cellular responses to Al3+ it is possible to discriminate between the Al-S and Al-R C. dactylon genotypes.