Klaus Brinkmann

Lead tolerance of Betula and Salix in the mining area of Mechernich/Germany

Natural populations of woody perennials on lead-mining sites in the Mechernich area of the Eifel Mountains were investigated with respect to soil factors determining the degree and type of heavy metal tolerance. Salix caprea L. (Goat Willow) grew on soils with up to 17000 mg kg−1 total lead (ca. 4000 mg kg−1 ammonium acetate-exchangeable Pb). Betula pendula Roth (Silver Birch) was found on soils containing as much as 29000 mg kg−1 total lead (7000 mg kg−1 ammonium acetate-exchangeable Pb). Other woody perennials, with the exception of the dwarf shrub Calluna vulgaris, were not found in the contaminated area even though they did occur in the immediate vicinity. The two lead-tolerant tree species did not form mixed populations.Because of a significantly lower Pb/Ca ratio in Salix soils (2.2) compared with Betula soils (7.4), a calcium-dependent mechanism of lead tolerance is suggested for Salix, but not for Betula.The Betula population could be divided into two groups, each showing a highly significant correlation between root-lead content and exchangeable lead amounts in the soil, but with different levels of lead uptake. The only soil factor distinguishing the two groups was found to be the level of soluble phosphate. A distinctly low level of soil phosphate correlated with a high lead concentration in roots of the one group (30000 mg Pb kg−1 DW), whereas high phosphate amounts corresponded with a much lower lead concentration in roots of the other (12000 mg Pb kg−1 DW−1). Since the correlation between lead in the soil and in plants was similar for the two groups, it is concluded that the type of lead tolerance in Betula is determined by the status of plant phosphate nutrition, rather than by simple phosphate precipitation in the soil.A comparison of growth between different populations of Betula seedlings on homogenized soils from the mining area revealed the Mechaernich population to be a distinct ecotype with respect to lead tolerance. The control population obtained from a non-contaminated area exhibited a lower degree of lead tolerance coupled with a two-step strategy of adaptation to lead.

Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions

SummaryThe sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)+ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.

Heavy metal tolerance in Festuca ovina L. from contaminated sites in the Eifel Mountains, Germany

Extremely high degrees of lead tolerance, measured by comparing rates of root extension in culture solutions, are reported from populations of Festuca ovina growing at two lead-mining sites (Westschacht and Keldenich-II) near Mechernich in the Eifel Mountains, Germany. Other populations from nearby heavy metal-contaminated areas show a considerably smaller degree of lead tolerance. Samples of Festuca ovina collected in the field at Westschacht and Keldenich-II contain higher levels of lead in their aerial organs than do those from other lead-contaminated sites. The main soil factor determining the high degree of lead tolerance is the high Pb/Ca ratio. Populations from soils with a low Pb/Ca ratio display a very low degree of tolerance. It is therefore concluded that in Westschacht and Keldenich-II plants, a genuine intracellular tolerance mechanism is present, allowing the accumulation of lead in aerial organs.Leaf samples of zinc-tolerant Festuca contain higher levels of zinc than do samples of non-tolerant plants. Lead and zinc amounts in leaves are correlated with the soil ratios of Pb/Ca and Zn/Ca, respectively, rather than with the absolute soil-metal levels.In a slightly lead-tolerant, but highly zinc-tolerant clone of Festuca ovina from a site contaminated with large amounts of lead and zinc (Plombières), lead was found to be the major factor affecting the inhibition of root extension with combined treatments of lead and zinc in culture solutions. In the highly lead-tolerant, zinc-sensitive population from Westschacht, zinc governs the response of root growth to combinations of the two metals. The results are discussed in terms of discriminating distinct types of heavy-metal tolerance.

Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the treeBetula pendula and identification of conserved protein regions

SummaryNitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch.

Welchen Informationswert haben in Rohextrakten gemessene Enzymaktivitäten für physiologische Probleme

SummaryUnder conditions simulating homogenisation and enzyme extraction from plant tissue, malate (1 mM) at pH=3.8 or 4.3 is drastically efficient in inactivating cristalline LDH. The enzyme is partially protected by higher concentrations of its substrate pyruvate.As concentrations of metabolic intermediates, including organic acids, are highly variable in plants, enzyme activities in crude extracts may be indicative of different denaturation rather than of the actual activity of any given enzyme.

Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria

Mitochondria from Vicia faba L. exhibit two pathways for the oxidation of endogenous NADH. One pathway is rotenone-sensitive and includes three phosphorylation sites, the other one is rotenone-insensitive and by-passes site I. This by-pass is located in the area of flavoproteins, but several lines of evidence suggest that the rotenone-insensitive oxidation of matrix-NADH is not mediated by the external NADH dehydrogenase. With saturating substrate concentrations the two pathways are superimposed in state 3 while in state 4 the electrons are transferred only via the by-pass. The rotenone-resistant electron flux is obviously regulated from the substrate side. We suggest that the function of the by-pass is to regulate the NADH/NAD+ ratio of the matrix space.

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.

Effect of Temperature on the Pathways of NADH-Oxidation in Broad-Bean Mitochondria

Abstract1. Respiration rates of broad-bean (Vicia faba) mitochondria were studied as a function of temperature. Arrhenius plots of all membrane-bound enzymes, as obtained with saturating substrate concentrations, revealed a break in the lower temperature range. That break was considered to indicate a phase transition of membrane phospholipids, characteristic for chilling-sensitive plants. A second discontinuity at 30°C occurred only with activities linked to energy conservation. — 2. The activation energies for the oxidation of NAD+-linked substrates differ between states 3 and 4. State 3 respiration of NAD+-linked substrates is the result a superimposition of two branches of electron transport, which can be separated by different sensibilities to rotenone. A characteristic temperature dependency of the respiratory control, as well as a shift of the low temperature break in the Arrhenius plot toward a higher temperature after state 4 to state 3 transition, are calculated to be caused by the superimposition of the two branches. — 3. The temperature dependency of the oxidation of extra-mitochondrial NADH and of succinate differs remarkably from that of the oxidation of matrix-NADH. It has been concluded that the rotenone-resistant oxidation of matrix-NADH and the oxidation of external NADH are mediated via different pathways with individual regulation sites.

Circadian rhythm in the kinetics of acid denaturation of cell membranes of Euglena gracilis.

SummaryThe denaturation of Euglena gracilis in aggressive acids has been investigated by means of the chlorophyll oxidation. A comparative study in different halogen acetic acids revealed that the reaction kinetics are determined by a reaction between the acid anion and the outer membrane. The denaturation of cell membranes in trichloracetic acid obeys clear second-order kinetics with respect to the overall acid concentration and approaches first-order kinetics with respect to the proton concentration. Osmotic shrinkage immediately increases the acid permeability of the membrane; the increased permeability persists some time after reversal of the osmotic reaction. In nondividing cells which are entrained by a 12:12h light:dark rhythm a strong daily fluctuation is seen in the energy of activation of the denaturation ranging between 20 kcal/M early in the day and 34 kcal/M early at night. Under continuous bright light the fluctuation continuous for at least one further cycle. The susceptibility of the membrane fluctuates synchronously with the motoric activity of the cells but approximately 180° apart from the susceptibility of the circadian clock to phase shifting signals.

Multiple periodicities in the circadian system of unicellular algae

Abstract Three periodicities in the circadian range are observed when measuring circadian parameters of unicellular organisms in long running experiments (more than 15 days). This is demonstrated for different organisms (Chlamydomonas, Euglena, Chlorella) and different parameters (auto kinesis, extracellular pH, CO2- and O2-partial pressure). Having excluded analytical and experimental artefacts (i.e. filter leakage and subpopulation effects), the multiple periodicities have to be interpreted in a physiological model. The three periodicities always exhibit two common features: The locations of the side peaks are symmetrical to the middle peak and their energy contribution is always the same. We therefore favour the model of multiplicative coupling between the circadian oscillator and a low frequency oscillator modulating the amplitude of the circadian rhythm. Since the low frequency rhythm is not correlated to any exogenously running periodicity of the experimental surroundings, it is considered as generated by an endogenous oscillator. This shows the existence of different biological long time oscillators in one single cell and contradicts the so-called master-clock hypothesis stating that one cell has only one clock related oscillator.