Roman Przymusiński

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.

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.

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.

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90–4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.

Organospecific responses of lupin seedlings to lead. I. Localization of lead ions and stress proteins

A tissue-printing technique was used to follow distribution of lead ions in different organs of lupin seedling with the histological localization of pathogenesis-related proteins designated as PR-L1 to PR-L6, which were found to be induced in lupin roots by heavy metals (Przymusiński and Gwóźdź 1999). Lead nitrate solution was supplied to the root tips and the histological distribution of the metal in lupin organs was visualized by staining with 0.6 % (w/v) of sodium rhodizonate. As the distance from the site of lead application increased, the amount of free lead ions decreased and in the petioles the metal was not detected at all. Lead ions were localized mostly in vascular bundles, which suggests that it was transported into the upper parts of seedlings with the transpiration stream.Immunohistochemical analysis of the tissue prints showed that as compared to the control lead visibly increased the accumulation of the PR proteins in roots, hypocotyls, stems and leaf petioles of the lupin seedling.The histological distribution of the PR protein differs from that of lead, and was localized in parenchymatic cells of root cortex, hypocotyl and stem. It is worth noticing that the stress protein was also observed in the leaf petioles where lead was not detected. This fact as well as marked enhancement of PR (L1–L6) proteins accumulation in lead treated seedlings and our earlier studies (Przymusiński and Gwóźdź 1994, 1999, Przymusiński et al. 1995) suggests that these proteins could be elements of plant’s defence system against both biotic and abiotic stressing factors.

Increased accumulation of the 16 × 103Mr polypeptide in lupin roots exposed to lead, copper and nitrite ions

Abstract The effects of lead, copper, sodium nitrite and indolo 3-acetic acid (IAA) on growth and protein patterns of roots of Lupinus luteus were investigated. All tested compounds affected root development inhibiting growth and causing thickening. Analysis of polypeptide patterns by SDS-polyacrylamide gel electrophoresis revealed that in the presence of heavy metal ions and NaNO2, an increased accumulation of a 16 × 103Mr polypeptide occurred. However, the increased quantity of this component cannot be directly connected with root growth limiting factors as it has not been found among polypeptides from IAA-treated roots. We postulate that the 16 × 103Mr polypeptide belongs to a group of non-specific stress proteins, which accumulate in the presence of toxic substances.

Cadmium affects microtubule organization and post-translational modifications of tubulin in seedlings of soybean (Glycine max L.)

Cadmium (Cd) is a non-essential heavy metal, toxic to all living organisms. The microtubule (MT) cytoskeleton appears to be one of the main targets of Cd action. In this study we present, with the use of various immunological approaches, the effect of Cd at moderate (85 μM) and high (170 μM) concentrations on the structure and functioning of the MT cytoskeleton in the root cells of soybean seedlings. As the result of heavy metal action, root growth was significantly diminished and was accompanied by a reduction in mitotic activity and disturbance in the structure of the MT arrays, including randomization of the cortical MT arrangement, distorted mitotic arrays and complete depolymerization of the MTs. Biochemical analysis revealed decreased levels of various α- and β-tubulin isoforms with a parallel down-regulation of most examined α-tubulin genes. Simultaneously, Cd treatment led to differentiated changes in the level of tubulin post-translational modifications, including tyrosination, detyrosination, acetylation, and polyglutamylation. Decreased tyrosination and polyglutamylation of particular tubulin isoforms accompanied by increase in the level of specific detyrosinated and acetylated isoforms implies augmented stability and reduced turnover of the MTs during stress conditions. Taken together, the obtained results indicate the significant impact of Cd on gene expression levels and subsequent post-translational processing of tubulin, which may be related to the impairment of MT cytoskeleton functioning in root cells.

Immunohisto- and cytochemical localization of PR-10 proteins induced by heavy metals in lupine roots

Most of the data concerning PR-10 proteins accumulation in plants under various environmental stress conditions are derived from biochemical analysis of transcript expression or polypeptide accumulation. In the current study, we examined the localization of PR (L1–L6) proteins, belonging to the PR-10 class family, in the root tips of yellow lupine seedlings using histo- and cytochemical methods. Immunohistochemical localization revealed the presence of PR-proteins in all examined yellow lupine root tip tissue, and lead treatment did not change the distribution of proteins in the roots. However, the metal induced a significant increase in the accumulation of PR-proteins in the meristematic cells and procambium tissue. An immunocytochemical approach with immunogold method demonstrated the cytoplasmic nature of the PR-proteins and their increased accumulation after copper treatment. The results confirmed the affiliation of PR (L1–L6) to PR-10 class family proteins, as well as their intracellular localization.