Władysław Polcyn

Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp. (Lupinus)

Induction, energy gain, effect on growth, and interaction of nitrate and nitrite reduction of Bradyrhizobium sp. (Lupinus) USDA 3045 were characterized. Both nitrate and nitrite were reduced in air, although nitrite reduction was insensitive to ammonium inhibition. Anaerobic reduction of both ions was shown to be linked with energy conservation. A dissimilatory ammonification process was detected, which has not been reported in rhizobia so far. Nevertheless, anaerobic conversion of nitrate to ammonium was lower than 40%, which suggests the presence of an additional, nitrite reductase of denitrifying type. Nitrite toxicity caused a non-linear relationship between biomass produced and >2 mM concentrations of each N oxyanion consumed. At > or =5 mM initial concentrations of nitrate, a stoichiometric nitrite accumulation occurred and nitrite remained in the medium. This suggests an inhibition of nitrite reductase activity by nitrate, presumably due to competition with nitrate reductase for electron donors. Lowering of growth temperature almost completely diminished nitrite accumulation and enabled consumption as high as 10 mM nitrate, which confirms such a conclusion.

Functional similarities of nitrate reductase from yellow lupine bacteroids to bacterial denitrification systems

Summary The nitrate-reducing activity of root nodules of field-grown yellow lupine plants ( Lupinus luteus cv. Ventus) was found to be about 25 and 100 times higher than that of roots and leaves, respectively. At least 97 percnt; of nodule nitrate reductase (NR) activity proved to be of bacteroidal origin. Data from the present work indicates that nitrate reductase (EC 1. 7. 99. 4) of symbiotic Bradyrhizobium sp. ( Lupinus ) cells possess some functional similarities to bacterial denitrification systems. The enzyme occurs in association with membranes, and electron transport from NADH to nitrate is dependent on the mediation of membrane components. Another feature in common with bacterial nitrate dissimilation is inhibition of bacteroidal nitrate reductase activity under aerobic conditions. Membrane permeabilization significantly alleviated this inhibitory effect. It was found that enzyme activity is not nitrate-dependent and peaks at pH 5.5-6.5. Rotenone inhibition indicated that NADH-quinol oxidoreductase, not succinate dehydrogenase, is the major participant in a very efficient succinate-dependent nitrate reductase activity. Pyruvate and malate were also highly effective in nitrate reduction. By contrast, oxaloacetate and 2-oxoglutarate were not efficient, even in combination with pyruvate, which suggests that this effect could not be caused by acetyl-SCoA limitation. These results suggest that a large part of the TCA cycle may not be necessary for nitrate reduction in yellow lupine bacteroids. The possible alternative pathways, which could provide electrons from pyruvate and malate to nitrate reductase, are discussed.

Dissimilatory Nitrate Reductase from Bradyrhizobium sp. (Lupinus): Subcellular Location, Catalytic Properties, and Characterization of the Active Enzyme Forms

Subcellular location, chlorate specificity, and sensitivity to micromolar concentrations of azide suggest that most of the anaerobically induced nitrate reductase (NR) activity in Bradyrhizobium sp. (Lupinus) could be ascribed to the membrane type of bacterial dissimilatory NRs. Two active complexes of the enzyme, NRI of 140 kDa and NRII of 190 kDa, were detected in membranes of the nitrate-respiring USDA strain 3045. Both enzyme forms were purified to homogeneity. Obtained specific antibodies showed that these native species were immunologically closely related and composed of largely similar 126-kDa, 65-kDa, and 25-kDa subunits. The finding that NRI and NRII share common epitopes suggests that they may not be different species, but rather two forms of the same enzyme.

Metabolism of amino acids in germinating yellow lupin seeds. II. Pathway of conversion of aspartate to alanine during the imbibition

The incorporation of 14C-aspartate during the imbibition of yellow lupin seeds resulted in the production of 14C-alanine and 14CO2. On the basis of tracer and enzymatic assays, conducted in vitro on the extract obtained from lupin seeds, it is postulated that aspartate can be converted to oxaloacetate, then, by phosphoenolopyruvate and pyruvate to alanine. This pathway can be catalyzed by the following enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, pyruvate kinase and alanine aminotransferase.

Changes in the activity of phosphoenolpyruvate carboxylating enzymes in germinating yellow lupin seeds

The rate of phosphoenolpyruvate carboxylation by extracts from germinating lupin seeds was measured through the H14CO3 fixation. PEP carboxylation in seed axes increased during their imbibition, mainly as a result of the increase in the activity of PEP carboxylase [EC 4.1.1.31]. However, the activity of PEP carboxykinase [EC 4.1.1.38], present during the first 3 hours of imbibition, as well as the activity of PEP-carboxykinase [EC 4.1.1.49], after 24 hours of imbibition, have also been shown. Possible physiological role of the changes in the activity of PEP carboxylases during lupin seeds germination is discussed.

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (Lupinus luteus)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.

Physio-Genetic Dissection of Dark-Induced Leaf Senescence and Timing Its Reversal in Barley

The organelle-specific physiology in and gene medleys during stress-induced barley leaf senescence are reversible prior to terminal programmed cell death phase. Barley crop model was analyzed for early and late events during the dark-induced leaf senescence (DILS) as well as for deciphering critical time limit for reversal of the senescence process. Chlorophyll fluorescence vitality index Rfd was determined as the earliest parameter that correlated well with the cessation of photosynthesis prior to microautophagy symptoms, initiation of DNA degradation, and severalfold increase in the endonuclease BNUC1. DILS was found characterized by up-regulation of processes that enable recycling of degraded macromolecules and metabolites, including increased NH4+ remobilization, gluconeogenesis, glycolysis, and partial up-regulation of glyoxylate and tricarboxylate acid cycles. The most evident differences in gene medleys between DILS and developmental senescence included hormone-activated signaling pathways, lipid catabolic processes, carbohydrate metabolic processes, low-affinity ammonia remobilization, and RNA methylation. The mega-autophagy symptoms were apparent much later, specifically on day 10 of DILS, when disruption of organelles—nucleus and mitochondria —became evident. Also, during this latter-stage programmed cell death processes, namely, shrinking of the protoplast, tonoplast interruption, and vacuole breakdown, chromatin condensation, more DNA fragmentation, and disintegration of the cell membrane were prominent. Reversal of DILS by re-exposure of the plants from dark to light was possible until but not later than day 7 of dark exposure and was accompanied by regained photosynthesis, increase in chlorophyll, and reversal of Rfd, despite activation of macro-autophagy-related genes.