Edward A. Gwóźdź

The message of nitric oxide in cadmium challenged plants

During the last decade it has been found that cadmium (Cd), one of the most toxic elements occurring in polluted environments, interferes with nitric oxide (NO), a multifunctional signaling molecule in living organisms. The formation of NO has been demonstrated in vivo in various plant tissues exposed to Cd stress, but unfortunately, the time and intensity of NO generation, relatively frequently shows conflicting data. What is more, there is still limited information regarding the functional role of endogenously produced NO in plants challenged with heavy metals. The first pharmacological approaches revealed that exogenously applied NO can alleviate cadmium toxicity in plants, promoting the direct scavenging of reactive oxygen species (ROS) or activating antioxidant enzymes. However, recent reports have indicated that NO even contributes to Cd toxicity by promoting Cd uptake and participates in metal-induced reduction of root growth. In view of this heterogeneous knowledge, much more puzzling if we consider results first obtained using exogenous NO sources, this review is focused mainly on the implication of endogenous NO in plant response to Cd exposure. Furthermore, a basic draft for NO mode of action during cadmium stress is proposed.

Plant cell responses to heavy metals: molecular and physiological aspects

The effect of lead, cadmium and cooper on protein pattern, free radicals and antioxidant enzymes in root of Lupinus luteus L. were investigated. Heavy metals inhibited growth of lupin roots, which was accompanied by increased synthesis and accumulation of a 16 kDa polypeptide (Przymusiński et al. 1991 Biochem. Physiol. Pflanzen., 187:51–57). This component has been earlier identified as immunologically related to Cu,Zn-superoxide dismutase (Przymusiński et al. 1995 Env.Exp.Bot., 35:485–495). However, more detailed study revealed that this stress-stimulated protein is composed of four to six polypeptides of different electrophoretic mobility. The most abundant polypeptides of the 16kDa region were found to be closely homologous to pathogen related proteins. The number and intensity of these polypeptides was highly variable in roots of individual seedlings, which suggests that they might represent separate allelic forms.Electron paramagnetic spectra revealed that at low lead concentrations the amplitude of the first derivative was similar to the control and distinctly increased at higher metal concentrations. On the other hand, at the lower lead concentrations the activity of antioxidant enzymes increased, whereas at higher metal doses the enzyme activities did not raise further (SOD) or even dropped (CAT, APOX). This implies that the responses of antioxidant system to lead is dose-dependent stimulated by low metal concentrations, whereas at the higher metal level the free radical emission is beyond the quenching capacity of antioxidant enzymes, which in turn might contribute to the reduced root growth.The effect of various heavy metals: Pb2+, Cd2+ and Cu2+ on phytochelatins and antioxidant enzymes depends on the kind of metal ion. Pb2+ and Cd2+ stimulated the PCs formation whereas Cu2+ was not effective. On the other hand, in root exposed to Cu the activity of catalase (CAT) was the highest as was the production of H2O2. The strong oxidative effect of Cu2+ ions which were not complexed by PCs suggests that these peptides might by involved in the cellular defense system by binding excessive heavy metal ions.On the basis of our results it can be concluded that in lupin roots exposed to heavy metals there is a complex defense system against metal phytotoxicity, which comprises of specific proteins, antioxidant enzymes and phytochelatins.

Nitric oxide implication in cadmium-induced programmed cell death in roots and signaling response of yellow lupine plants.

The sequence of events leading to the programmed cell death (PCD) induced by heavy metals in plants is still the object of extensive investigation. In this study we showed that roots of 3-day old yellow lupine (Lupinus luteus L.) seedlings exposed to cadmium (Cd, 89μM CdCl(2)) resulted in PCD starting from 24h of stress duration, which was evidenced by TUNEL-positive reaction. Cd-induced PCD was preceded by a relatively early burst of nitric oxide (NO) localized mainly in the root tips. Above changes were accompanied by the NADPH-oxidase-dependent superoxide anion (O(2)(·-)) production. However, the concomitant high level of both NO and O(2)(·-) at the 24th h of Cd exposure did not provoke an enhanced peroxynitrite formation. The treatment with the NADPH-oxidase inhibitor and NO-scavenger significantly reduced O(2)(·-) and NO production, respectively, as well as diminished the pool of cells undergoing PCD. The obtained data indicate that boosted NO and O(2)(·-) production is required for Cd-induced PCD in lupine roots. Moreover, we found that in roots of 14-day old lupine plants the NO-dependent Cd-induced PCD was correlated with the enhanced level of the post-stress signals in leaves, including distal NO cross-talk with hydrogen peroxide.

Effects of exogenous nitric oxide on the antioxidant capacity of cadmium-treated soybean cell suspension

Nitric oxide (NO) is a small, ubiquitous molecule, whose physiological function in plants has recently been widely investigated. It seems that one of its pivotal properties is the antioxidant capacity, enabling plants to alleviate the effects of the oxidized stress. In this work we investigated the role of NO in soybean (Glycine max L. Cv. Navico) cell suspension treated with cadmium. Sodium nitroprusside (SNP), nitric oxide donor, markedly decreased the negative influence of Cd2+ on cell growth. It was also found to stimulate superoxide dismutase (SOD, EC 1.15.1.1). Using specific fluorochromes — dihydroethidine (DHE) and 2′,7′- dichlorofluorescein (DCFH-DA) it was shown that NO was very effective in reducing the level of superoxide anion (O2·−) and hydrogen peroxide, respectively. Furthermore, as evaluated by means of NO specific fluorochrome 4,5-diaminofluorescein diacetate (DAF-2DA), increased production of NO was found in Cd-treated cells. In cadmium-stressed cells SNP lowered the level of oxidized proteins.Our results suggest that the antioxidant properties of nitric oxide in Cd-treated soybean cells rely mainly on its ability to direct scavenging of ROS and stimulation of the antioxidant system.

The stress-stimulated 16 kDa polypeptide from lupin roots has properties of cytosolic Cu:Zn-superoxide dismutase

An increased accumulation of 16 kDa polypeptide was found in lupin roots exposed to lead, copper and nitrite ions [Przymusinski R. and Gwoźdź E. A. (1994) Env. Exp. Bot. 34, 63–68].(24) The 16 kDa component was isolated from lead-treated lupin roots, purified by DEAE-Sephacel chromatography and electroeluted after polyacrylamide gel electrophoresis. The pure protein was used to produce antibody; the 16 kDa polypeptide specifically cross-reacted with its own antiserum as well as with antibody against Cu:Zn-superoxide dismutase (SOD) from spinach seeds. By two-dimensional SDS-PAGE, the 16 kDa polypeptide was resolved into a few electromorphs of intense cross-reaction with the anti-SOD serum, which differs in quantity and position from the stained subcomponents. Native polyacrylamide gel electrophoresis followed by specific staining revealed four bands of SOD activity, which was the highest in purified and crude extracts of lead treated roots, respectively. Electrophoresis of extracts from roots exposed to various stressing factors followed by electroblotting on nitrocellulose membrane and immunodetection with antibodies either against lupin 16 kDa polypeptide or spinach Cu:Zn-SOD showed that both antisera specifically reacted with the 16 kDa region in all variants; however, the strongest cross-reaction was observed with heavy metals and nitrite. The results obtained by ‘Western blotting’ were confirmed by photochemically assayed SOD activity, which was higher in extracts of roots treated with heavy metal, nitrite and sulfite ions or exposed to salt stress. However, the highest SOD activity was found in root extracts enriched in the 16 kDa polypeptide. It was also found that the antibody against the 16 kDa polypeptide markedly decreased the SOD activity in lupin root extracts. Thus, we provide evidence that the heretofore unidentified lupin ‘stress protein’ that accumulates at high rates in roots treated with various stressors and toxic ions can be classified as cytosolic Cu:Zn-SOD. It appears that there are common responses of plant cells to a diversity of environmental stressors that enhance free radical production, which in turn stimulates the production of superoxide dismutase, the main scavenger of highly reactive oxygen species.

Inorganic lead changes growth and polypeptide pattern of lupin roots

Summary Cultivation of seedlings of Lupinus luteus L. in the presence of increasing lead concentrations resulted in inhibited root growth and reduced mitotic activity. Lead also decreased the quantity of soluble proteins in roots as well as affected the polypeptide pattern in this fraction by rising the relative amounts of polypeptides of 16, 34 and 68 kDA in the gels. The effect of lead was most pronounced for the polypeptide of 16 kDa. In vivo pulse labelling of roots followed by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that Pb 2+ quickly and selectively promotes the synthesis of some polypeptides, whereas the production of others was reduced.

Regeneration of enzymatically isolated protoplasts of the moss Funaria hygrometrica L. Sibth.

Abstract Protoplasts from protonemata of the moss Funaria hygrometrica were obtained by one-step enzymatic degradation of the cell walls. A pre-incubation with a non-ionic detergent Tween 80 and the use of a hemicellulase in combination with cellulase and Macerozyme appeared to be important for successful protoplast isolation. Protoplasts were cultured in hanging drops of a mineral nutrient medium supplied with 0.3 M mannitol and 0.5% glucose. The presence of glucose was required for survival and the subsequent division of protoplasts. Cultured protoplasts regenerated cell walls within 3 days of isolation and developed into multi-cellular protonemata after one month of culture.

Organospecific responses of lupin seedlings to lead Localization of hydrogen peroxide and peroxidase activity

In an earlier work using tissue printing method, we found that the PR-10 stress protein was observed in leaf petiole of lupin seedling where lead was not detected (Przymusiński et al. 2001). These results suggested the presence of substance(s) mediating a signal transduction from directly affected cells to distant organs. As the hydrogen peroxide was found to be involved in signal transduction pathway, in the present paper, we analysed the level of H2O2 in the organ of lupin seedlings exposed to Pb2+ with spectrophotometric method and tissue printing technique. It was unequivocally demonstrated that the level of H2O2 and the activity of peroxidase increased in every tested organ of lead-treated lupin seedling. Both the level of H2O2 and the activity of POX were correlated with amount of Pb2+ ions in the cells (Przymusiński et al. 2001) and decreased in tissues more and more distant from the site of metal application. On the other hand, there was no correlation between the histological localization of H2O2 and peroxidase. Our results seem to confirm the hypothesis that H2O2 may act as a signalling substance involved in the induction of PR protein synthesis. It was indicated that there is high degree of correlation between the localization of H2O2 and the histological localization of PR-10 proteins (Przymusiński et al. 2001) in every tested organ of lupin seedling. The presented hypothesis is also supported by the fact that H2O2 and PR-10 proteins are detected in organs and tissues where Pb2+ was not found at all.

Selection in vitro and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (Cucumis sativus)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 μM cadmium chloride (CdCl2) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.

The effect of 3-Indolylacetic acid on the differentiation of plastids in callus culture ofCichorium intybus L.

SummaryThe effect of 3-indolylacetic acid (IAA) on structure of plastids in cells of callus developing on the phloem explants of chicory roots was investigated. In the absence of IAA the proplastids in the initial expiant developed into typical chloroplasts. The presence of increasing IAA concentrations in the medium resulted in a gradual reduction of the thylakoid system accompanied by an increasing starch content of the plastids. Depending on the IAA concentration used, various types of plastids from typical chloroplasts to typical amyloplasts were found. A possible relationship between auxins and sugar metabolism is indicated.