Fate of 13C in metabolic pathways and effects of high CO2 on the alteration of metabolites in Rumex obtusifolius L.

In present study, in vivo turn-over of ¹³CO₂ to organic acids such as oxalate and citrate in Rumex obtusifolius L. was explored. Conversion of fixed carbon to oxalate was studies using “new leaves”, i.e., leaves removed from 2-month-old-plants grown under different environmental conditions. Collected new leaves and stems were subjected to metabolomic analyses using capillary electrophoresis mass spectrometry. The results showed the mobilization of metabolites from stems to new leaves, where active TCA cycle and oxalate pathways occurred. The ¹³C labeling experiments also indicated that these pathways are active in new leaves. Subsequently, we compared the effects of high carbon dioxide level (1000 ppm) and nutrients (Hoagland’s formulation) on the metabolite accumulation in R. obtusifolius. Data analysed by both principal component and hierarchical clustering analyses revealed significant changes in metabolite accumulation. The accumulation of most abundant metabolite oxalate in leaves was affected by both high CO₂ as the carbon source and nutrients. We suggest that the common weed R. obtusifolius may proliferate in cultivated lands under high CO₂ level, a potential cause of global warming.     

Targeted metabolomics in an intrusive weed, <Emphasis Type="Italic">Rumex obtusifolius</Emphasis> L., grown under different environmental conditions reveals alterations of organ related metabolite pathway

This study was intended to analyze the metabolic pathway of Rumex obtusifolius L. (Broad-leaved dock), destructive weeds worldwide, in relation to major environmental factors (light and temperature). It was found that R. obtusifolius can be classified as plants in accumulating major organic acids such as oxalate in leaves and citrate in stems (Miyagi et al., Metabolomics 6:146–155 2010). The organ specific accumulation of certain metabolites was dissected by metabolomics approach in relation to metabolic pathway. Light or dark experiments showed that in the case of the oxalate accumulation, the major or the most dominated pathway was found to be the citrate-isocitrate-oxalate shunt. Furthermore, under the dark and/or low temperature (5°C) leaves showed sustainable growth with normal accumulation of TCA metabolites. Unlike leaves, there was a different pattern of metabolite accumulation in stems. Other metabolites such as amino acids also showed the organ specific alterations under the different ambient environments.     

Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Organic acids differ in enhancing phosphorus uptake by Triticum aestivum L.—effects of rhizosphere concentration and counterion

Organic acids such as citrate and oxalate have been implicated in enhancing many rhizosphere processes including nutrient acquisition. This study was conducted to determine the importance of organic acid type and concentration on rhizosphere P mobilization and subsequent uptake by wheat (Triticum aestivum L.) roots and its translocation to shoots. A single wheat plant was grown in soil-filled rhizosphere microcosms and allowed to pass through a KH₂PO₄ ³³P-isotopically labeled patch of calcareous soil. Two days after ³³P-injection, citrate and oxalate at concentrations of 1 mM and 10 mM were injected into the microcosms at the same patch every day over a period of 4 days. Oxalate resulted in a several-fold enhancement in plant ³³P accumulation, while citrate had no such effect. In comparison with oxalate, high rates of citrate mineralization were observed suggesting that this reduced its potential to enhance plant ³³P acquisition. This study concludes that organic acids cause an increase in P mobilization and P uptake by wheat but that this response is highly organic acid specific.     

Distribution and chemical speciation of aluminum in the Al accumulator plant,Melastoma malabathricum L.

The Al accumulation mechanisms in an Al accumulator plant, Melastoma malabathricum L. (Melastoma), was investigated. Al was located in the upper epidermal cells and also distributed in mesophyll cells in leaf sections. In root sections, Al was found in all the root tissues, particularly in the epidermis and endodermis. Al concentrations in young leaves, mature leaves, old leaves, and roots were 8.0, 9.2, 14.4, and 10.1 mg g¹, respectively. Approximately 45% of total Al in oldest leaves, and approximately 60% of total Al in leaves of other positions and roots were extracted in Tris-HCl buffer (pH 7.0). Since Al in the residual parts was mostly dissolved in hot 0.5 M H₂SO₄ containing 2% cetyl trimethylammonium bromide, residual Al seemed to consist mainly of monomeric Al and Al bound to pectic substances and hemicellulose. Al in the Tris-HCl extract consisted of non-monomeric Al (complexed form). Oxalate concentration in the Tris-HCl extract in leaves was significantly higher in the +Al treatment than in the –Al treatment and there was a positive correlation between the Al concentration and oxalate concentration. ²⁷Al NMR spectrum of fresh leaves indicated the presence in the order of monomeric Al, Al-oxalate, Al-(oxalate)₂, and Al-(oxalate)₃ in intact leaves.     

Inhibition of isocitrate lyase from Pseudomonas indigofera by itaconate

The effect of the inhibitor itaconate on the activity of purified isocitrate lyase from Pseudomonas indigofera was examined for the reaction in both directions. Itaconate was found to equilibrate very slowly with its enzyme-bound form, so that a rapid change in itaconate concentration produced a gradual change in reaction velocity which eventually reached a new steady state. Kinetic studies of this relaxation phenomenon indicated that itaconate inhibited by binding the enzyme only after prior binding of glyoxylate, thus mimicking the kinetic behavior of succinate. On the basis of these studies, the dissociation constants for itaconate and glyoxylate from their respective enzyme-bound forms were calculated. More than half of the isocitrate lyase was complexed by glyoxylate during cleavage of saturating isocitrate. The rate constant for release of itaconate from the enzyme was calculated to be about 0.2 min^?1. Direct binding of [¹⁴C]itaconate and [¹⁴C]succinate to isocitrate lyase at pH 6.8 was measured. Some binding of both ligands was found in the absence of glyoxylate, which was stimulated by the presence of 1 mm glyoxylate. These results suggest that there are up to three or more binding sites per active subunit, but that only one of these is catalytic.     

Isolation and properties of watermelon isocitrate lyase

The glyoxylate cycle enzyme, isocitrate lyase (EC 4.1.3.1) was purified from cotyledons of Citrullus vulgaris (watermelon). The final preparation, which had been 97-fold purified with a specific activity of 16.1 units/mg protein in a yield of 36%, was homogeneous by gel- and immunoelectrophoretic criteria. The tetrameric enzyme had: a molecular weight of 277 000, a sedimentation coefficient of 12.4 s, and a K_m for D_s-isocitrate equal to 0.25 mM. Isocitrate lyase from this source is not a glycoprotein as shown by total carbohydrate content after precipitation by trichloroacetic acid of the purified enzyme. Reduction of the enzyme with thiols increased activity and maximal activity was obtained with at least 5 mM dithiothreitol. EDTA partially substituted for thiol in freshly isolated enzyme. Watermelon isocitrate lyase was also protected against thermal denaturation at 60° for at least 1 hr by 5 mM Mg²⁺ plus 5 mM oxalate. Oxalate was a competitive inhibitor with respect to isocitrate (K_i: 1.5 μM, pH 7.5, 30°).     

Aluminium Uptake and Translocation in Al Hyperaccumulator Rumex obtusifolius Is Affected by Low-Molecular-Weight Organic Acids Content and Soil pH

Background and Aims

High Al resistance of Rumex obtusifolius together with its ability to accumulate Al has never been studied in weakly acidic conditions (pH > 5.8) and is not sufficiently described in real soil conditions. The potential elucidation of the role of organic acids in plant can explain the Al tolerance mechanism.

Methods

We established a pot experiment with R. obtusifolius planted in slightly acidic and alkaline soils. For the manipulation of Al availability, both soils were untreated and treated by lime and superphosphate. We determined mobile Al concentrations in soils and concentrations of Al and organic acids in organs.

Results

Al availability correlated positively to the extraction of organic acids (citric acid < oxalic acid) in soils. Monovalent Al cations were the most abundant mobile Al forms with positive charge in soils. Liming and superphosphate application were ambiguous measures for changing Al mobility in soils. Elevated transport of total Al from belowground organs into leaves was recorded in both lime-treated soils and in superphosphate-treated alkaline soil as a result of sufficient amount of Ca available from soil solution as well as from superphosphate that can probably modify distribution of total Al in R. obtusifolius as a representative of “oxalate plants.” The highest concentrations of Al and organic acids were recorded in the leaves, followed by the stem and belowground organ infusions.

Conclusions

In alkaline soil, R. obtusifolius is an Al-hyperaccumulator with the highest concentrations of oxalate in leaves, of malate in stems, and of citrate in belowground organs. These organic acids form strong complexes with Al that can play a key role in internal Al tolerance but the used methods did not allow us to distinguish the proportion of total Al-organic complexes to the free organic acids.     

Caenorhabditis elegans and Ascaris suum: inhibition of isocitrate lyase by itaconate

The largest forms of isocitrate lyase from Caenorhabditis elegans and Ascaris suum of 543,000 and 549,000 daltons, respectively, can be purified from three- to five-fold in excellent yield by pelleting from extracts at 160,000g for 4 hr. Isocitrate lyase in the pellet is much more stable toward proteolysis. Itaconate which both inhibits isocitrate lyase and suppresses the level of this enzyme in bacteria inhibits the partially purified isocitrate lyase from both C. elegans and A. suum. The inhibition is noncompetitive with respect to d_s-isocitrate at one itaconate concentration. The K_i values at 30 C, pH 7.7, are 19 and 7.3 μM for the enzyme from C. elegans and A. suum, respectively. Itaconate inhibits the growth of C. elegans in random axenic as well as monoxenic cultures. At a concentration of 10 mM, itaconate is more effective in the inhibition of random axenic cultures than is oxalate, maleate, or succinate. At 60 mM itaconate, reproduction of C. elegans larvae is completely abolished.     

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP⁺) [threo-d_s-isocitrate:NADP⁺ oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH₂COO^?→acetylCoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml^?1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP⁺) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.     

Mechanisms of citrate transport and exchange in corn mitochondria

Previous work (Birnberg, Jayroe, Hanson 1982 Plant Physiol 70: 511-516) demonstrated that corn mitochondria (Zea mays L.) can accumulate citrate by a malate- and phosphate-independent proton symporter. This uptake and symport of other ions were investigated. Passive swelling experiments indicated that corn mitochondria can accumulate several other anions by proton symport, but only isocitrate is taken up nearly as effectively as citrate. At the optimal pH (4.5), active uptake of carrier-free [¹⁴C]citrate in 50 micromolar mersalyl is inhibited by fourteen anions, but only the I₅₀ (the concentration of inhibitor required to reduce uptake of carrier-free [¹⁴C]citrate by 50%) values of citrate (0.08 millimolar) and d-and l-isocitrate (0.5 millimolar) are less than 4 millimolar. Isocitrate is a competitive inhibitor of citrate uptake and [¹⁴C]isocitrate is accumulated with a K_m similar similar to its I₅₀. Valinomycin reduces net active citrate accumulation at pH 7.5, consistent with the relatively low V_max for citrate uptake. At pH 4.5, mersalyl reduces the rate of citrate uptake without changing the affinity of the carrier for citrate. Thus, the corn mitochondria have a high-affinity, mersalyl-insensitive carrier selective for citrate that also transports isocitrate.     

不同因子对荞麦中草酸含量的影响

用不同化合物从根部喂养麦幼苗,测定其根叶中草酸含量的变化。结果表明：异柠檬酸、抗坏血酸及其前体物均可不同程度地降低荞麦根叶中草酸含量;而乙醇酸与乙醛酸则显著提高其草酸含量,表明荞麦叶片草酸合成主要来自乙醇酸途径,而非来自抗坏血酸等途径。水培条件下,以铵态氮或尿素等作唯一氮源时,荞麦中草酸含量远低于以硝态氮培养的;将谷氨酸或丝氨酸加到含硝态氮培养液中也能显著降低其草酸含量,不同氮素影响荞麦草酸含量可能与乙醇酸途径有关。     

Itaconate controls its own synthesis via feedback-inhibition of reverse TCA cycle activity at IDH2

Macrophages undergo extensive metabolic reprogramming during classical pro-inflammatory polarization (M1-like). The accumulation of itaconate has been recognized as both a consequence and mediator of the inflammatory response. In this study we first examined the specific functions of itaconate inside fractionated mitochondria. We show that M1 macrophages produce itaconate de novo via aconitase decarboxylase 1 (ACOD1) inside mitochondria. The carbon for this reaction is not only supplied by oxidative TCA cycling, but also through the reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase (IDH). While macrophages are capable of sustaining a certain degree of itaconate production during hypoxia by augmenting the activity of IDH-dependent reductive carboxylation, we demonstrate that sufficient itaconate synthesis requires a balance of reductive and oxidative TCA cycle metabolism in mouse macrophages. In comparison, human macrophages increase itaconate accumulation under hypoxic conditions by augmenting reductive carboxylation activity. We further demonstrated that itaconate attenuates reductive carboxylation at IDH2, restricting its own production and the accumulation of the immunomodulatory metabolites citrate and 2-hydroxyglutarate. In line with this, reductive carboxylation is enhanced in ACOD1-depleted macrophages. Mechanistically, the inhibition of IDH2 by itaconate is linked to the alteration of the mitochondrial NADP⁺/NADPH ratio and competitive succinate dehydrogenase inhibition. Taken together, our findings extend the current model of TCA cycle reprogramming during pro-inflammatory macrophage activation and identified novel regulatory properties of itaconate.     

Ecophysiologic investigations in the family of the Mesembryanthemaceae

Summary Overnight accumulation of malate, citrate, and isocitrate in a large number of species of Mesembryanthemaceae grown under identical environmental conditions was studied. Of the 27 species investigated, 24 showed malate accumulation, which in 3 cases was accompanied by considerable overnight accumulation of citrate. In the leaves of the same plants, the Na⁺, K⁺, Cl^-, SO₄ ^2-, and PO₄ ^3- contents were determined. Although the plants were not exposed to substrates of high NaCl content, they exhibited extraordinarily high levels of Na⁺ and Cl^-. All plants accumulated, much more Na⁺ than K⁺. No readily discernible correlation between the amount of any particular ion and the extend of CAM was found. It is concluded that halophilism and CAM are widespread phenomena in the family of Mesembryanthemaceae that possess ecologic significance.Abbreviations CAM Crassulacean Acid Metabolism - PEP Phosphoenolpyruvate On leave from Canberra     

Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (pK_a's of 6.9 and 8.6). The pH dependence of the pK_i for succinate, a product of isocitrate cleavage, implied that a dissociable group (pK_a of 6.0) on the enzyme functions in binding succinate. The pK_i's for maleate and itaconate (succinate analogs) were similarly pH dependent. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pK_i's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pK_m for isocitrate and the pK_i for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (pK_a of 5.8). The pH dependence of the pK_i for homoisocitrate was similar. In addition the K_i for succinate and K_m for isocitrate were dependent upon Mg²⁺ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pK_a of 5.8) and one on phosphoenolpyruvate (pK_a of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate.     

Oxalate- and Glyoxylate-Dependent Growth and Acetogenesis by Clostridium thermoaceticum

The acetogenic bacterium Clostridium thermoaceticum ATCC 39073 grew at the expense of the two-carbon substrates oxalate and glyoxylate. Other two-carbon substrates (acetaldehyde, acetate, ethanol, ethylene glycol, glycolaldehyde, glycolate, and glyoxal) were not growth supportive. Growth increased linearly with increasing substrate concentrations up to 45 mM oxalate and glyoxylate, and supplemental CO₂ was not required for growth. Oxalate and glyoxylate yielded 4.9 and 9.4 g, respectively, of cell biomass (dry weight) per mol of substrate utilized. Acetate was the major reduced end product recovered from oxalate and glyoxylate cultures. ¹⁴C labeling studies showed that oxalate was subject to decarboxylation, and product analysis indicated that oxalate was utilized by the following reaction: 4^-OOC-COO^- + 5H₂O → CH₃COO^- + 6HCO₃^- + OH^-. Oxalate- and glyoxylate-dependent growth produced lower acetate concentrations per unit of cell biomass synthesized than did H₂-, CO-, methanol-, formate-, O-methyl-, or glucose-dependent growth. Protein profiles of oxalate-grown cells were dissimilar from protein profiles of glyoxylate-, CO-, or formate-grown cells, suggesting induction of new proteins for the utilization of oxalate. C. thermoaceticum DSM 2955 and Clostridium thermoautotrophicum JW 701/3 also grew at the expense of oxalate and glyoxylate. However, oxalate and glyoxylate did not support the growth of C. thermoaceticum OMD (a nonautotrophic strain) or six other species of acetogenic bacteria tested.     

Biogeochemistry of oxalate in the antarctic cryptoendolithic lichen-dominated community

Cryptoendolithic (hidden in rock) lichen-dominated microbial communities from the Ross Desert of Antarctica were shown to produce oxalate (oxalic acid). Oxalate increased mineral dissolution, which provides nutrients, creates characteristic weathering patterns, and may ultimately influence the biological residence time of the community. Oxalate was the only organic acid detectable by HPLC, and its presence was verified by GC/MS. Community photosynthetic metabolism was involved in oxalate production since rates of ¹⁴C-oxalate production from ¹⁴C0₂ were higher in light than in dark incubations. Flaking of the sandstone at the level of the lichen-dominated zone a few millimeters beneath the rock surface can be explained by dissolution of the sandstone cement, which was enhanced by Si, Fe, and Al oxalate complex formation. Added oxalate was observed to increase the solubility of Si, Fe, Al, P, and K. Oxalate's ability to form soluble trivalent metal-oxalate complexes correlated with the observed order of metal oxide depletion from the lichen-dominated zone (Mn > Fe > Al). Thermodynamic calculations predict that Fe oxalate complex formation mobilizes amorphous Fe oxides (ferrihydrite) in the lichen-dominated zone, and where oxalate is depleted, ferrihydrite should precipitate. Hematite, a more crystalline Fe oxide, should remain solid at in situ oxalate concentrations. Oxalate was not a carbon source for the indigenous heterotrophs, but the microbiota were involved in oxalate mineralization to CO₂, since oxalate mineralization was reduced in poisoned incubations. Photooxidation of oxalate to C02 coupled with photoreduction of Fe(Ill) may be responsible for oxalate removal in situ, since rates of ¹⁴C-oxalate mineralization in dark incubations were at least 50% lower than those in the light. Removal of oxalate from Si, Fe, and Al complexes should allow free dissolved Si, Fe, and Al to precipitate as amorphous silicates and metal oxides. This may explain increased siliceous crust (rock varnish or desert varnish) formation near the surface of colonized rocks were light intensity is greatest.Offprint requests to: C.G. Johnston.     

Aconitate decarboxylase 1 participates in the control of pulmonary Brucella infection in mice

Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1^-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1^-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.     

Soil chemical properties affect the concentration of elements (N,P, K,Ca, Mg,As, Cd,Cr, Cu,Fe, Mn,Ni, Pb,and Zn) and their distribution between organs of Rumex obtusifolius

Background and aims

The ionome (elemental composition) of grassland species has rarely been studied at the level of individual organs and little is known about effects of soil chemical properties on the ionome. Using the model oxalate plant Rumex obtusifolius, we asked how its biomass production and the distribution of elements between its organs is affected by soil chemical properties.

Methods

We established a pot experiment with R. obtusifolius planted in acidic non-contaminated control and in slightly acidic and alkaline soils anthropogenically contaminated by the risk elements As, Cd, Pb, and Zn. Both contaminated soils were untreated and treated by lime and superphosphate. We determined biomass production and the concentrations of elements in its organs.

Results

Biomass production was negatively related to the mobility of micro- and risk elements. Restricted transport of micro- and risk elements from belowground organs into leaves was recorded in untreated contaminated soils. In both lime-treated soils and in superphosphate-treated alkaline soil, elevated transport of micro- and risk elements from belowground organs into leaves was recorded in comparison to untreated contaminated soils. The lowest concentrations of micro- and risk elements were recorded in stems and seeds, followed by belowground organs and leaves.

Conclusions

R. obtusifolius is an As-, Cd-, Pb-, and Zn-excluder and is sensitive to high availability of micro- and risk elements in the soil. Soil chemical properties affect the distribution of essential elements within the plant greatly.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.