Cytotoxic aldehyde generation in heart following acute iron-loading

Although the mechanism of myocardial failure following acute iron poisoning is not known, excess iron-catalyzed free radical generation is conjectured to play a role. The effects of time (0 to 360 minutes) on total iron concentrations, glutathione peroxidase activity, and cytotoxic aldehyde production in heart of mice (B6D2F1, n = 65) were first investigated following acute iron-loading (20 mg iron dextran i.p./mouse). In a subsequent experiment, the effects of dose (0 to 80 mg iron dextran i.p./mouse, n = 75) on the aforementioned parameters were investigated. Our results show that the concentrations of cytotoxic aldehydes: (1) significantly differ over-time, with corresponding increases in total concentrations of iron (r = 0.93, p < 0.001); and (2) increase parallel to the total dose of iron administered (r = 0.95, p < 0.001). Furthermore, dose-and time-dependent alterations to glutathione peroxidase activity are observed, which is most likely due to an acute up-regulation of the enzyme as an endogenous protective response to increased free radical activity in the heart subsequent to iron-loading. While no single mechanism is likely to account for the complex pathophysiology of acute iron-induced heart failure, our results shown that iron-loading can result in significant free radical generation, as quantified by cytotoxic aldehydes, in heart tissue of mice. This is the first report on the effects of time and dose on cytotoxic aldehyde generation and glutathione peroxidase activity in heart of mice following acute iron-loading.     

Potato purple top phytoplasma‐induced disruption of gibberellin homeostasis in tomato plants

Phytoplasmas are phloem‐inhabiting, cell wall‐less bacteria that cause numerous plant diseases worldwide. Plants infected by phytoplasmas often exhibit various symptoms indicative of hormonal imbalance. In this study, we investigated the effects of potato purple top (PPT) phytoplasma infection on gibberellin homeostasis in tomato plants. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellic acid (GA₃). The decrease in GA₃ content in diseased plants was correlated with down regulation of genes responsible for biosynthesis of bioactive GAs ( GA20ox1 and GA3ox1) and genes involved in formation of GA precursors [geranyl diphosphate synthase (GPS) and copalyldiphosphate synthase (CPS)]. Exogenous application of GA₃ at 200 µmol L^?1 was able to restore the GA content in infected plants to levels comparable to those in healthy controls, and to attenuate the characteristic ‘big bud’ symptoms induced by the phytoplasma. The interesting observation that PPT phytoplasma‐infected plants had prolonged low expression of key GA biosynthesis genes GA20ox1 and GA3ox1 under GA deficiency conditions led us to hypothesise that there was a diminished sensitivity of the GA metabolism feedback regulation, especially GA biosynthesis negative feedback regulation, in those affected plants, and such diminished sensitization in early stages of infection may represent a central element of the phytoplasma‐induced disruption of GA homeostasis and pathogenesis.     

Characteristics of Fe‐deficiency‐induced acidification in subterranean clover

Iron-deficiency-induced acidification is one of the important reactions of plant Fe-deficiency-stress response, but the overall understanding of this reaction is limited. The characteristics of Fe-deficiency-induced acidification of subterranean clover (subclover) (Trifolium brachycalycinum Katzn. and Morley cv. Koala) were studied in this paper. Plants were grown hydroponically under -Fe conditions, and Fe-deficiency-induced acidification was determined using pH-stat, back-titration and chemical equilibrium procedures. Fe-deficiency-induced acidification was undetectable during the first day after Fe-deficiency stress initiation, but the maximum acidification rate was attained by the second day, when plants exhibited visual chlorosis symptoms. The acidification rate was relatively constant with increasing Fe-deficiency chlorosis, suggesting that a critical level of Fe deficiency was needed to trigger acidification, but that once the acidification process was initiated, the intensity of acidification was independent of severity of Fe deficiency. Net H⁺-release (PR) rate determined using a chemical equilibrium method and net acidity release (AR) rate determined using a back-titration method were practically identical, indicating that Fe-deficiency-induced acidification involved almost entirely the release of free H⁺, not organic acid. In the assay temperature range of 5 to 35°C, PR rate was highest at about 20°C. Net acidity release rate was almost totally inhibited at pH values ≤4.5 and increased with increasing assay pH up to pH 9. The pH effect occurred within 30 min of incubation initiation, implying that the effect of pH is probably on the activity of H⁺ transport through the plasma membrane, not on the quantity of responsible protein(s). Cations were required in the incubation solution for Fe-deficiency-induced acidification. Divalent cations in the assay solution resulted in a higher AR rate than monovalent cations, and essential cations resulted in a higher AR rate than non-essential cations, indicating that the relative effectiveness of cations is related to the efficiency of their absorption by plant roots. These results are discussed in relation to their practical significance and the mechanisms of Fe-deficiency-induced acidification.     

Water relations and growth of tomato fruit pericarp tissue

The water relations of young tomato fruit pericarp tissue were examined and related to tissue expansion. The relationship between bulk turgor pressure and tissue expansion (as change in fresh mass or length of tissue) was determined in slices of pericarp cut from young, growing fruit by incubation in different osmotic concentrations of polyethylene glycol 6000 or mannitol. The bulk turgor of this tissue was low (about 0.2 MPa), even in fruit from plants that were otherwise fully turgid, whether measured psychrometrically or by length change in osmotic solutions. The rate of tissue growth at maximum turgor was less than that at moderate turgor unless calcium was added to the incubation medium. However, added calcium also decreased the rate of growth at lower turgor pressures. Yield turgor was < 0.1 MPa, but it was increased by the addition of calcium ions. Electrolyte leakage from tissue was greatest at maximum turgor pressure but was decreased by the addition of calcium ions or osmoticum. Tissue growth was unaffected by a range of plant growth regulators (IAA, abscisic acid, benzyladenine and GA₃) but was inhibited, particularly at high turgor, by low concentrations of malic or citric acid. The low turgor pressure of pericarp tissue could be due to the presence of apoplastic solutes within the pericarp, and evidence for this is discussed. Thus, fruit tissue may be able to maintain optimal expansion rates only at moderate turgor and low calcium concentration.     

Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens

The moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel D5- and D6- desaturases are required. To isolate one of these, PCR-based cloning was used, and resulted in the isolation of a full-length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N-terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b₅-related region and a C-terminal sequence with low similarity (27% identity) to acyl-lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ-linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a D6-desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC–MS. Only the transformed yeast cells were able to introduce a further double bond into the D6-position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.     

Multiple forms of ADP‐glucose pyrophosphorylase from tomato leaf

ADP‐glucose pyrophosphorylase (AGP, EC 2.7.7.27) was partially purified from tomato ( Lycopersicon esculentum Mill. cv. Laura) leaf by a procedure previously used for purification of AGP from tomato pericarp. SDS‐PAGE and western blot analysis of the final preparation indicated that the leaf enzyme is composed of two subunits of 50 and 54 kDa. Two‐dimensional PAGE and western blot analysis of the same preparation, however, revealed at least six isoforms of the large subunit and two isoforms of the small subunit. The leaf AGP is very sensitive to regulation by 3‐phosphoglycerate and inorganic phosphate. Its properties are compared to those of AGP from tomato fruit.     

Effect of silver ions on ethylene biosynthesis by tomato fruit tissue

Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver-free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC ± silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC.     

Inactivation by gene disruption of 2‐cysteine‐peroxiredoxin in Synechocystis sp. PCC 6803 leads to increased stress sensitivity

2‐Cysteine‐peroxiredoxins (2‐CP) constitute a ubiquitous group of enzymes which reduce toxic alkyl hydroperoxides. In higher plants it was shown that the nuclear encoded 2‐CPs are posttranslationally imported into the chloroplasts, the site of most active oxidative metabolism in plants (Baier and Dietz 1997, Plant J. 12; 179‐190). The genome of the bluegreen alga Synechocystis (EMBL acc. # D64000) encodes a 2‐CP which shares 60% homology to higher plant 2‐CPs on the gene level and about 70% on the level of the mature protein. In order to elucidate the physiological significance of 2‐CPs for photosynthetic organisms, the 2‐CP gene was mutated in Synechocystis sp. PCC 6803 by insertion of a kanamycin gene cartridge. Following complete segregation mutant lines were analyzed for growth and photosynthetic parameters. The mutants revealed decreased growth rates as compared to the wild type. Growth inhibition was relieved after lowering the concentration of Fe or trace elements in the growth medium. Chlorophyll a fluorescence transients as induced by saturating light pulses were used as indicator for the state of photosynthesis. The effective quantum yield decreased at lower light intensities in the mutants as compared to the wild type Synechocystis . Simultaneously, electron transport rates saturated at lower light intensities in the mutants. These data provide the first evidence that 2‐CPs play a pivotal protective role in photosynthesis.     

Free radical involvement in the generation of trans‐root potential

The identity of the naturally occurring compounds that accept electrons from plasma membrane-bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical-generating and -quenching systems on the trans-root electrical potential, which had previously been shown to be coupled to plasma membrane-bound redox systems. The material was the primary root of 8-day-old maize (Zea mays L.) seedlings. Trans-root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans-root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans-root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane-bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Stress‐induced changes in polyamine and tyramine levels can regulate proline accumulation in tomato leaf discs treated with sodium chloride

The effect of salt stress on proline (Pro) accumulation and its relationship with the changes occurring at the level of polyamine (PA) metabolism and tyramine were investigated in leaf discs of tomato (Lycopersicon esculentum). The rate of accumulation of Pro, PA and tyramine was higher in the salt-sensitive than in the salt-tolerant cultivar. In the salt-sensitive cultivar, Pro started to accumulate 4 h after the onset of the NaCl treatment, its maximum level being reached 27 h later. The lag phase was associated with a rapid decrease in putrescine (Put) and spermidine (Spd) and some increase in 1,3-diaminopropane (Dap), a product of Spd and/or spermine (Spm) oxidation. This was followed by an increase in agmatine (Agm), cadaverine (Cad), Spm and tyramine. α-DL-difluoromethylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), induced a decrease in the Put level in both control and stressed discs, while α-DL-difluoromethylomithine (DFMO), an inhibitor of ornithine decarboxylase (ODC, EC 4.1.1.17), caused a decrease in Spd and Spm levels only in salinized discs. These data suggest that ADC is operating under both control and stress conditions, whereas ODC activity is promoted only in response to salt stress. DFMA also depressed the salt-induced Pro accumulation while DFMO did not inhibit this response. In salt-stressed leaf discs, the decrease in Spd level in response to methylglyoxal-bis-(guanylhydrazone) (MGBG) or cyclohexylammonium (CHA) treatment suggests that salt stress did not block SAM decarboxylase or Spd synthase activities. However, the increased level of Dap reflected a salt stress-promoted oxidation of PA. CHA and MGBG had no effect on Pro accumulation. Putrescine, Dap and especially tyramine supplied at low concentrations stimulated the Pro response which was, however, suppressed by application of Spm. Treatment with aminoguanidine, an inhibitor of diamine oxidases, also strongly inhibited Pro accumulation. These data suggest that salt-induced Pro accumulation in tomato leaf discs is closely related to changes in their PA metabolism, either via substrate-product relationships or regulatory effects at target(s) which remain to be characterized.