Changes in Phosphorus Fractions and Phytase Activity of Rice Seeds during Germination

The present experiment provides information on the phosphorus compounds in rice seeds and elucidates the changes they undergo during germination. In ungerminated seeds, the bulk of total-P appears in phytin (about 76 per cent). It is then dephosphorylated in course of germination with a simultaneous accumulation of large amounts of inorganic-P. Lipid-P increases very rapidly from 0 to 24 hours. The increase up to 72 hours is gradual, after which it drops at 96 hours and again rises to a maximum after 120 hours. The ester-P and RNA-P, fractions increase in concentration with time of germination (except 120 hours). Protein-P begins to fall after 48 hours, while DNA-P remains more or less constant throughout the experiment. The two pH optima recorded for phytase activity at 4.0 and 9.0, suggests that there exist two phytases, one acidic and the other alkaline. Both behave similarly during germination with a continuous increase throughout the course of the experiment. The enzyme with an optimal pH at 4 hydrolyses phytin more actively than the other with the pH optimum at 9.0. Phytase shows maximum activity at a stage when most of the phytin has disappeared; the metabolic significance is uncertain.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles.

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48-144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual "noncristate" mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of "noncristate" mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Germination of Echinochloa crus-galli (Barnyard Grass) Seeds under Anaerobic Conditions : Respiration and Response to Metabolic Inhibitors

Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO₂ in Echinochloa and rice is lower and the peak rate of CO₂ evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO₂] N₂/[CO₂] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.

Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N₂, but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed.

     

The effect of nitrite on cytochrome oxidase

Nitrite inhibits the oxygen uptake by the system ferrocytochrome c-cytochrome oxidase with Ki = 1.5 mM. In the absence of ferrocytochrome c the oxygen uptake by cytochrome oxidase in the presence of nitrite was observed indicating that the enzyme has some nitrite oxidase activity. Nitrite induces changes in optical difference spectra of cytochrome oxidase and, in particular, the formation of the transient band at 607 nm. The reciprocal relation was observed between the intensity of this band and the rate of the oxygen uptake by cytochrome oxidase. This means that the form of the enzyme with this band does not involved in the nitrite oxidase activity. It is suggested that the nitrite oxidase activity relates to the oxygen binding site rather than the cytochrome c binding site of the enzyme.     

Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa.

The germination of conidiospores of wild-type Neurospora crassa was found to be dependent upon the function of the cytochrome-mediated electron transport pathway. The cyanide-insensitive alternate oxidase did not contribute significantly to the respiration of these germinating spores. The dormant spores contained all of the cytochrome components and a catalytically active cytochrome c oxidase required for the activity of the standard respiratory pathway, and these preserved components were responsible for the accelerating rates of oxygen uptake which began immediately upon suspension of the spores in an incubation medium. Mitochondria of the dormant spores contained all of the subunit peptides of the functional cytochrome c oxidase; nevertheless, de novo synthesis of these subunits began at low rates in the first stages of germination. Reactivation of the respiratory system of germinating N. crassa spores seems not to be dependent initially upon the function of either the mitochondrial or cytoplasmic protein-synthesizing systems. The respiratory activity of spores of three mutant cytochrome c oxidase-deficient strains of N. crassa also was found to depend upon the function of the cytochrome electron transport pathway; the dormant and germinating spores of these strains contained a catalytically active cytochrome c oxidase. Cytochrome c oxidase may be present in the dormant and germinating spores of these strains as the result of a developmental-phase-specific synthesis of and requirement for the enzyme.     

Ultrastructural changes of organelles in coleoptile cells during anaerobic germination of rice seeds

Summary Ultrastructural changes of organelles, especially those of mitochondria in rice seedlings germinated under strictly anaerobic conditions were investigated.The embryos of dry seeds had slightly modified mitochondria, characterized by an electron transparent matrix with few cristae and electron opaque patches. These mitochondria developed normally for 48 hours irrespective of whether oxygen was present or not. However, after 72 hours' germination under anaerobic conditions vesiculation of the cristae developed and progressed greatly for the subsequent 24 hours and most of the spaces in the mitochondrion became occupied with vacuolated cristae. Vesiculation seemed to be the effect of cessation of energy supply from the mitochondria themselves.Ultrastructural changes of other organelles such as the plastids and endoplasmic reticulum were also observed after 96 hours under anaerobic conditions.     

Effect of magnetic field on activity of cytochrome oxidase not moved or moved relative to magnetic field lines

The influence of constant homogeneous magnetic fields (700 to 13,000 gauss) on cytochrome c oxidase activity as a function of strength and duration of magnetic field and as a function of enzyme concentration at 0-5 degrees C in solution was measured on samples not moved or moved relative to the magnetic field lines. Increases or decreases of enzyme activity were observed depending on whether or not the samples were so moved. These changes persisted for hours after terminating exposure to the magnetic field.     

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inhibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48–144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual “noncristate” mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of “noncristate” mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

彩绒革盖菌多酚氧化酶活性研究

本文测定了彩绒革盖菌在PDY液体培养基中的多酚氧化酶活性。在30℃,110 r／min,恒温振荡培养条件下,多酚氧化酶第14天达产酶高峰,最高酶活549.Ou;酶作用的最适酸碱度为pH 5.0;最适作用温度30℃;Mn2+、Ba2+、Mg2+等离子对多酚氧化酶有激活作用,Ag+、Fe3+等离子对酶活则有明显的抑制作用。     

Fractional Analyses of Sterols in Soybean Harvested in Japan

Debranching enzyme activity in rice seeds increased during the early stage of ripening and then decreased, and increased again during germination. The inactive enzyme accumulated rapidly in ripening seeds from the 20th day after flowering.

Radioactive amino acids were readily incorporated into the active debranching enzyme after their absorption into immature rice seeds. Subsequently, the radioactivity increased in the inactive enzyme, accompanying a decrease in the active enzyme. We concluded that the debranching enzyme in rice seeds is synthesized during ripening in active form and that it accumulates in inactive form, which can be reactivated during germination.     

Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.     

Regulation of alternative oxidase activity in higher plants

Plant mitochondria contain two terminal oxidases: cytochrome oxidase and the cyanideinsensitive alternative oxidase. Electron partioning between the two pathways is regulated by the redox poise of the ubiquinone pool and the activation state of the alternative oxidase. The alternative oxidase appears to exist as a dimer which is active in the reduced, noncovalently linked form and inactive when in the oxidized, covalently linked form. Reduction of the oxidase in isolated tobacco mitochondria occurs upon oxidation of isocitrate or malate and may be mediated by matrix NAD(P)H. The activity of the reduced oxidase is governed by certain other organic acids, notably pyruvate, which appear to interact directly with the enzyme. Pyruvate alters the interaction between the alternative oxidase and ubiquinol so that the oxidase becomes active at much lower levels of ubiquinol and competes with the cytochrome pathway for electrons. These requirements for activation of the alternative oxidase constitute a sophisticated feed-forward control mechanism which determines the extent to which electrons are directed away from the energy-conserving cytochrome pathway to the non-energy conserving alternative oxidase. Such a mechanism fits well with the proposed role of the alternative oxidase as a protective enzyme which prevents over-reduction of the cytochrome chain and fermentation of accumulated pyruvate.     

Excess boron reduces polyphenol oxidase activities in embryo and endosperm of maize seed during germination

The effects of increasing concentrations of boron (0, 0.1, 1, 10 and 20 mM) as boric acid on the rate of germination and polyphenol oxidase activities in embryo and endosperm tissues of maize seeds (Zea mays L. cv. Arifiye) were studied. The germination percentage of maize seeds was not affected by boron concentrations up to 10 mM, and decreased by 20 mM. Distilled water and lower boron concentrations (0.1 and 1 mM) increased polyphenol oxidase activities at the beginning of germination up to 12 h whereas its excess levels (10 and 20 mM) decreased polyphenol oxidase activities in embryos and endosperm during germination. Polyphenol oxidase activities with o-diphenolic substrates (caffeic acid, catechol and dopa) were found to be higher than with a monophenolic substrat (tyrosine) in both embryos and endosperms. Further, caffeic acid oxidizing polyphenol oxidase was found to show more activity in embryos of the seeds germinating in distilled water when compared to other substrates.     

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice.

The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.     

甲基磺酸乙酯(EMS)对水稻离体幼穗影响的初步研究

用不同浓度的EMS处理水稻离体幼穗,测定培养初期的呼吸强度、细胞色素氧化酶活性和同工酶以及苹果酸脱氢酶同工酶,发现低浓度的EMS对细胞呼吸强度以及细胞色素氧化酶活性有刺激作用,而高浓度的EMS则表现为抑制效应。呼吸强度和酶活性与幼穗培养力之间具有平行关系。研究结果表明,EMS的原初生理效应是影响幼穗细胞的代谢过程,幼穗体细胞诱导频率的改变是幼穗培养初期供能系统受到影响的结果。     

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice

Summary The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2–6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.