Reversible interconversion of the functional state of the gene regulator FNR from Escherichia coli in vivo by O2 and iron availability

FNR, the gene regulator of anaerobic respiratory genes of Escherichia coli is converted in vivo by O₂ and by chelating agents to an inactive state. The interconversion process was studied in vivo in a strain with temperature controlled synthesis of FNR by measuring the expression of the frd (fumarate reductase) operon and the reactivity of FNR with the alkylating agent iodoacetic acid. FNR from aerobic bacteria is, after arresting FNR synthesis and shifting to anaerobic conditions, able to activate frd expression and behaves in the alkylation assay like anaerobic FNR. After shift from anaerobic to aerobic conditions, FNR no longer activates the expression of frd and reacts similar to aerobic FNR in the alkylation assay. The conversion of aerobic (inactive) to anaerobic (active) FNR occurs in the presence of chloramphenicol, an inhibitor of protein synthesis. Anaerobic FNR can also be converted post-translationally to inactive, metal-depleted FNR by growing the bacteria in the presence of chelating agents. The reverse is also possible by incubating metal-depleted bacteria with Fe²⁺. From the experiments it is concluded that the aerobic and the metal-depleted form of FNR can be transferred post-translationally and reversibly to the anaerobic (active) form. The response of FNR to changes in O₂ supply therefore occurs at the FNR protein level in a reversible mode.Abbreviation BV_red = reduced benzyl viologen     

Indole-3-acetic acid and rice coleoptile elongation under anoxia

To investigate the presence of a possible synergistic effect of IAA and anaerobiosis on rice coleoptile elongation, excised coleoptiles grown in aerobic and anaerobic conditions were tested and compared with intact seedling aerial parts for response to exogenous IAA and for levels of endogenous IAA. Excised coleoptiles were fed with³H-IAA to study aerobic and anaerobic IAA metabolism. Our results can be summarized as follows. (1) IAA and anaerobiosis have no synergistic effect on rice coleoptile elongation. (2) This behavior is due not to an inhibition of IAA uptake but probably to a reduced and different IAA metabolism in coleoptile grown in the absence of oxygen. (3) In anaerobic rice coleoptiles, the conversion to inactive conjugate (IAA-Asp) could be adopted as means of detoxification in the case of abnormally high and unutilized IAA levels. (4) The increase in IAA level found in coleoptiles of intact seedlings during anaerobic treatment could be due, as in the roots, to a translocation from the endosperm, in which the hormone is contained in a great quantity.     

Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Anaerobic nitrate and ammonium metabolism in flood-tolerant rice coleoptiles

The tolerance of germinating rice seedlings to anaerobiosiscannot be fully accounted for by ethanolic fer mentation alone.Nitrate metabolism (nitrate reduction to NH plus its subsequentassimilation) may provide an additional sink mechanism for excessprotons and NADH produced during anaerobiosis. To follow thefate of nitrate, ¹⁵N-labelled nitrate and ammonium incorporationin aerobic and anaerobic rice coleoptiles was examined using¹⁵N-edited ¹H NMR and gas chromatography-mass spectrometry methods.After 22h of treatments, protein-free Ala, Glu, Gln, and     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.     

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.Abbreviations CHX cycloheximide - Fd ferredoxin - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase The authors wish to thank Dr. J. Turner (Rothamsted Experimental Station, Harpenden, UK) for providing Fd-GOGAT antibody and Dr. H. Sakakibara (Nagoya University, Nagoya, Japan) for Fd-GOGAT clone. This research was supported by the National Research Council of Italy, special project RAISA, sub-projekt N. 2, paper N. 2174.     

Exogenous Nitrate as a Terminal Acceptor of Electrons in Rice (Oryza sativa) Coleoptiles and Wheat (Triticum aestivum) Roots under Strict Anoxia

To elucidate the physiological role of exogenous nitrate under anaerobic conditions, we studied the effect of 10 mM KNO₃ on the mitochondrial ultrastructure in rice (Oryza sativa L.) coleoptiles and in wheat (Triticum aestivum L.) roots, detached from four-day-old seedlings, under strict anoxia. In wheat roots, following 6-h-long anoxia in the absence of exogenous nitrate, the mitochondrial membranes were partially degraded and, after 9 h under anoxia, the mitochondrial membranes and the membranes of other organelles were completely destroyed. In rice coleoptiles, the partial membrane degradation was observed only after 24 h and their complete breakdown after 48 h of anaerobiosis. In the presence of exogenous nitrate, no membrane destruction was noticed even after 9 and 48 h of anaerobiosis in wheat roots and rice coleoptiles, respectively. These results indicate that exogenous nitrate exerts protective action as a terminal electron acceptor, alternative to the molecular oxygen. Our findings are compared with the results of other researchers concerning the adverse or favorable nitrate action on plant growth, metabolism, and energy status under anaerobic stress.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

Effects of anoxia on mitochondrial biogenesis in rice shoots: modification of in organello translation characteristics

Shoots of germinating rice (Oryza sativa L.) seedlings are able to grow under anoxia and to withstand long periods of anoxic treatment. Mitochondria were purified from aerobically germinated and anaerobically treated rice shoots by differential and isopycnic centrifugation and were found to consist of two subpopulations. The mitochondrial subpopulation of higher density was used for further characterization. Ultrastructural studies showed anaerobic mitochondria to be significantly different from aerobic mitochondria, with a matrix of lower density and more developed cristae. Aerobic and anaerobic mitochondria also differed in their specific activities for fumarase and succinate dehydrogenase, which were significantly lower after the anoxic treatment. In vivo labeling of seedlings with l-[³⁵S]methionine and subsequent isolation of the mitochondria indicated that anoxia induced a drastic decrease, but not a total inactivation, of the synthesis of mitochondrial proteins. In organello protein synthesis showed that anaerobic mitochondria were able to synthesize most of the polypeptides synthesized by aerobic mitochondria, although only in the presence of exogenous ATP, as would occur under anoxia. Anaerobic mitochondria, but not aerobic mitochondria, could carry out protein synthesis without a functional respiratory chain. Thus, mitochondrial protein synthesis was found to be potentially functional in the rice shoot under anoxia.     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of ¹⁴CO₂ from tissues fed with [¹⁴C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.     

Control of gene expression by FNR-like proteins in facultatively anaerobic bacteria

Facultatively anaerobic bacteria are able to adapt to many different growth conditions. Their capability to change their metabolism optimally is often ensured by FNR-like proteins. The FNR protein ofEscherichia coli functions as the main regulator during the aerobic-to-anaerobic switch. Low oxygen tensions activate this protein which is expressed constitutively and is inactive under aerobic conditions. The active form is dimeric and contains a [4Fe−4S]²⁺ cluster. The direct dissociation of the cluster to the [2Fe−2S]²⁺ cluster by the effect of oxygen leads to destabilization of the FNR dimer and to loss of its activity. The active FNR induces the expression of many anaerobic genes; the set comprises over 100 of controlled genes. Many other bacteria contain one or more FNR analogues. All these proteins form the FNR family of regulatory proteins. Properties of these proteins are very distinct, sometimes even among representatives of different strains of the same bacterial species. FNR-like proteins together with other regulators (e.g., two-component system ArcBA, nitrate-sensing system NarXL,etc.) control a complicated network of modulons that is characteristic for every species or even strain and enables fine tuning of gene expression.     

Growth and energy status of arrowhead tubers,pondweed turions and rice seedlings under anoxic conditions

The responses of two aquatic plants, arrowhead (Sagittaria pygmaea Miq.) and pondweed (Potamogeton distinctus A. Benn), to anoxia were compared with those of rice (Oryza sativa L.). Shoot elongation of arrowhead tubers was enhanced at around 1 kPa O₂, whereas that of pondweed turions was slight in air and reached a maximum in the absence of O₂. Anaerobic enhancement of alcohol dehydrogenase (ADH) activity took place in rice coleoptiles but not in arrowhead and pondweed shoots. Shoots of both arrowhead and pondweed maintained a more stable energy status than did the rice coleoptile under anaerobic conditions. Total adenylate nucleotide contents of arrowhead and pondweed shoots were constant under anaerobic conditions. Adenylate energy charge in both shoots remained at a high and stable level of more than 0·8 for at least 8 d. Three forms of ADH from arrowhead shoots were separated by starch gel electrophoresis, showing that the activity of each ADH form was different under aerobic and anaerobic conditions. The incorporation of ³⁵S-labelled Cys and Met into soluble proteins in arrowhead shoots showed active protein biosynthesis and an involvement of a special set of polypeptides in the anaerobiosis.     

Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation

A 14.5 kDa protein with antigenic components in common with pea leaf ferredoxin was detected on transblots of the soluble proteins of pea root plastids. The amount of this protein was found to increase during the induction of nitrate assimilation in pea roots, reaching a maximal level at 8–12 h. Concurrent with this, a fourfold increase in NADPH-dependent ferredoxin-NADP⁺ oxidoreductase (FNR) activity was observed corresponding to an increase in the amount of this protein detected immunologically on transblots using a leaf FNR antibody. These changes were not observed in plastids from roots of plants grown on ammonia or depleted of nitrogen. It is suggested that in addition to the already well reported induction by nitrate of nitrate reductase and nitrite reductase, there is a co-induction of a plastid located ferredoxin and FNR. Both these proteins are necessary for the transfer of reductant generated by the oxidative pentose phosphate pathway to nitrite reductase.     

Patterns of polypeptide synthesis in various maize organs under anaerobiosis

The pattern of protein synthesis was compared in several organs of maize (Zea mays L.) under aerobic and anaerobic conditions. Protein synthesis was measured by [³⁵S]methionine incorporation and analysis by two-dimensional native-SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis and fluorography. The aerobic protein-synthesis profiles were very different for root, endosperm, scutellum and anther wall. However, except for some characteristic qualitative and quantitative differences, the patterns of protein synthesis during anaerobiosis were remarkably similar for these diverse organs and also for mesocotyl and coleoptile. The proteins synthesized were the anaerobic polypeptides (ANPs) which have been previously described in anaerobic roots of seedlings. Leaves exhibited no detectable protein synthesis under anaerobic conditions, and died after a short anaerobic treatment. Evidence is presented that the ANPs are not a generalized response to stress. This indicates that the ANPs are synthesized as a specific response to anaerobic conditions such as flooding.Abbreviations ADH alcohol dehydrogenase - ANP anaerobic polypeptide - SDS sodium lauryl sulfate     

Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings

Abstract Aerobically germinated seedlings of rice and Echinochloa were found to survive when placed in an anaerobic environment for 4 d, whereas pea and maize seedlings did not. Although root and shoot growth were inhibited in rice and Echinochloa under anaerobiosis, growth resumed when the seedlings were returned to aerobic conditions. Alcohol dehydrogenase (ADH) activity increased more, and protein synthesis was greater, in the shoots than in the roots under anaerobic conditions. These results suggest that, in anaerobiosis-tolerant species, ADH activity and protein synthesis in the shoots represents or results from metabolic adaptations to low oxygen. These results are discussed in terms of plant establishment and growth in a low-oxygen environment.