Alterations in Cd-induced gene expression under nitrogen deficiency in Hordeum vulgare.

The inter-relation between nitrogen availability and cadmium toxicity was studied in roots of barley seedlings with emphasis on the analysis of expression of 10 selected genes relevant for growth in the presence of toxic Cd concentrations. The response to Cd exposure differed quantitatively or qualitatively for the 10 genes in dependence of the N supply. Transcripts of glutathione synthase, glutathione reductase, glutathione peroxidase and dehydroascorbate reductase were measured as parameters involved in antioxidant defence, metallothionein, phosphoenolpyruvate carboxylase and phytochelatin synthase (PCS) were analysed as genes related to heavy metal binding, and vacuolar ATPase subunits VHA-E and VHA-c and a NRAMP-transporter as genes being implicated in Cd transport. Reprogramming of the Cd response was most obvious for PCS and NRAMP whose transcript levels were unaltered and down-regulated, respectively, in the presence of Cd at adequate N, but strongly up-regulated upon Cd exposure under conditions of nitrogen deficiency. Different responses to Cd at varying N supply were also seen for the antioxidant genes. The results on gene expression are discussed in context with the changes in biochemical parameters, and underline the importance of evaluating the general growth conditions of a plant when discussing its specific response to a stressor such as Cd. The sequence of the nramp cDNA was filed at the EMBL/GenBank/DDBJ Databases under the accession number AJ514946.

Tricarboxylic acid cycle enzymes of the ectomycorrhizal basidiomycete, Suillus bovinus.

In crude cell extracts of the ectomycorrhizal fungus, Suillus bovinus, activities of citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase have been proved and analyzed. Citrate synthase exhibited high affinities for both its substrates: oxaloacetate (Km = 0.018 mM) and acetyl-CoA (Km = 0.014 mM). Aconitase showed better affinity for isocitrate (Km = 0.62 mM) than for citrate (Km = 3.20 mM). Analysis of isocitrate dehydrogenase revealed only small maximum activity (60 nmol x mg protein(-1) x min(-1)), the enzyme being exclusively NADP+-dependent. Using the artificial electron acceptor dichlorophenol indophenol, activity and substrate affinity of succinate dehydrogenase were rather poor. Fumarase proved Fe2+-independent. Its affinity for malate was found higher (Km = 1.19 mM) than that for fumarate (Km = 2.09 mM). High total activity of malate dehydrogenase could be separated by native PAGE into a slowly running species of (mainly) cytosolic (about 80%) and a faster running species of (mainly) mitochondrial origin. Affinities for oxaloacetate of the two enzyme species were found identical within limits of significance (Km = 0.24 mM and 0.22 mM). The assumed cytosolic enzyme exhibited affinity for malate (Km = 5.77 mM) more than one order of magnitude lower than that for oxaloacetate. FPLC on superose 12 revealed only one activity band at a molecular mass of 100 +/- 15 kDa. Activities of 2-oxoglutarate dehydrogenase and of succinyl-CoA synthetase could not be found. Technical problems in their detection, but also existence of an incomplete tricarboxylic acid cycle are considered. Metabolite affinities, maximum activities and pH-dependences of fumarase and of malate dehydrogenase allow the assumption of a reductive instead of oxidative function of these enzymes in vivo.

Capacity of enzymes of the euphorbiacea Aleurites montana involved in CO2-fixation, compared to plants having C3-, C4- and Crassulacean acid metabolism.

Capacities of phosphoenolpyruvate carboxylase (PEP-Co), ribulose bisphosphate carboxylase (Rubisco), NADP+ malic enzyme (ME) and of malate dehydrogenase (MDH) were measured in the Euphorbiacea Aleurites montana, grown under 700 ppm CO2 for four weeks prior to enzyme extraction. For comparison Bryophyllum daigremontiana (CAM). Saccharum officinarum (C4) and Capsicum frutescens (C3) were treated in the same way. PEP-Co capacity of Aleurites was in the range of 12-, that of Capsicum approx. 26 nmol x min(-1) x mg protein(-1), without significant influence of the light period or CO2-treatment. In contrast, the activity of the enzyme from Saccharum was, depending on the duration of light, 160- respectively 96 times higher than that of the tung-oil tree. In Bryophyllum a rather low activity in the morning was increased during the day to approx. 230 nmol x min(-1) x mg protein(-1) in plants grown in the greenhouse and to approx. 115 nmol x min(-1) x mg protein(-1) in those from the growth chamber. Malate was hardly detectable in extracts of Aleurites, whereas it was high in Bryophyllum, depending on the light period. The ratio of average PEP-Co to Rub-Co capacity was high for the CAM-plant (20:1), somewhat lower for sugar cane (10:1), but almost at equality for Aleurites (0.9:1) and chilli (0.8:1). For the NADP+ malic enzyme, low capacity (20 to 28 nmol x min(-1) x mg protein(-1)) was found for Aleurites and for Capsicum, whereas it was 10 to 17 times higher in Saccharum. In Bryophyllum, the activity was up to 80 nmol x min(-1) x mg protein, dependent on light period. MDH capacity was extremely high in all plants investigated. Highest rates (10-20 micromol x min(-1) x mg protein(-1)), were obtained for Bryophyllum, followed by sugar cane and Capsicum with 5-8 micromol x min(-1) x mg protein(-1). Again, the lowest capacity was found in extracts of Aleurites with approx. 1.3 to 1.6 micromol x min(-1) x m protein(-1). Thus, in Aleurites montana no indication for C4- or Crassulacean acid metabolism was obtained. Therefore, the earlier observed very efficient uptake of CO2 cannot be explained by a high expression of the PEP-Co protein, known to occur in CAM- and C4-plants.

Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus.

Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.

Complete sequence of glycolytic enzymes in the mycorrhizal basidiomycete, Suillus bovinus.

Axenic cultures of Suillus bovinus were cultivated in inorganic liquid medium with glucose as a carbon source at 25 degrees C and continuous supply of oxygen by aeration with compressed air in the dark. Exogenous fructose as sole carbon source yielded about 50% less increase in dry weight than glucose. This resulted from different uptake velocities. Sucrose as sole exogenous carbon source yielded no measurable increase in dry weight. In glucose cultures, activities of all glycolytic enzymes were found. Maximum specific activities varied largely (from about 60 [fructose 6-phosphate kinase] to about 20,000 [triosephosphate isomerase] nmoles.mg protein-1.min-1). Apparent K(m)-values also varied over more than two orders of magnitude (0.035 mM [pyruvate kinase] to 6.16 mM [triosephosphate isomerase]). Fructose 6-phosphate kinase proved to be the fructose 2,6-bisphosphate-regulated type, aldolase the divalent cation-dependent (class II) type and glyceratephosphate mutase the glycerate 2,3-phosphate-independent type of the respective enzymes. Eight of the 10 enzymes exhibited pH-optima between 7.5-8.0. Triosephosphate isomerase and pyruvate kinase showed highest activities at pH 6.5. Regulatory sites within the glycolytic pathway of Suillus bovinus are discussed; fructose 6-phosphate kinase appears to be its main bottle neck.

In vitro photoinactivation of catalase isoforms from cotyledons of sunflower (Helianthus annuus L.).

Catalase (EC 1.11.1.6) isoforms CAT 2 through CAT 8 were purified from peroxisomes of sunflower (Helianthus annuus L.) cotyledons and photoinactivated in vitro. Action and absorbance spectra between 380 and 727 nm wavelength showed most prominent maxima at 405 nm suggesting an inactivation mediated by light absorption of heme groups. First order kinetics of inactivation were observed for CAT 6 through CAT 8 (isoform group B), which are composed of four 55-kDa subunits. Inactivation constants ki depended on photon fluence rates in the studied range between 8.3 and 660 microE m(-2) s(-1). The maximal value of ki was about 4.0 h(-1), corresponding to a half-life of about 10 min. Heme groups and 55-kDa apoprotein moieties of group B isoforms were degraded during irradiation, but both degradation processes occurred at rates lower than those of inactivation. Quantitative evaluations contradicted the view that photoinactivation was caused by destruction or dissociation of heme but suggested apoprotein damage leading to the loss of activity. Group A isoforms CAT 2 through CAT 5, containing both 55- and 59-kDa subunits, were less photosensitive than the isoforms of group B. In addition, irradiated group A isoforms reached a low plateau of residual activity, whereas group B isoforms were inactivated completely. The 59-kDa subunits in group A isoforms were much more resistant to photodegradation than the 55-kDa subunits of group B isoforms and also much more resistant than their own 55-kDa cosubunits. Results presented here are compared with catalase photoinactivation and turnover in vivo and discussed with respect to a physiological significance of catalase isoforms in plant peroxisomes.