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Developmental Studies on Microbodies in Wheat Leaves : I. Conditions Influencing Enzyme Development

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Abstract

Catalase, glycolate oxidase, and hydroxypyruvate reductase, enzymes which are located in the microbodies of leaves, show different developmental patterns in the shoots of wheat seedlings. Catalase and hydroxypyruvate reductase are already present in the shoots of ungerminated seeds. Glycolate oxidase appears later. All three enzymes develop in the dark, but glycolate oxidase and hydroxypyruvate reductase have only low activities. On exposure of the seedlings to continuous white light (14.8 x 10(3) ergs cm(-2) sec(-1)), the activity of catalase is doubled, and glycolate oxidase and hydroxypyruvate reductase activities increase by 4- to 7-fold. Under a higher light intensity, the activities of all three enzymes are considerably further increased. The activities of other enzymes (cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase) are unchanged or only slightly influenced by light. After transfer of etiolated seedlings to white light, the induced increase of total catalase activity shows a much longer lag-phase than that of glycolate oxidase and hydroxypyruvate reductase. It is concluded that the light-induced increases of the microbody enzymes are due to enzyme synthesis. The light effect on the microbody enzymes is independent of chlorophyll formation or the concomitant development of functional chloroplasts. Short repeated light exposures which do not lead to greening are very effective. High activities of glycolate oxidase and hydroxypyruvate reductase develop in the presence of 3-amino-1,2,4-triazole which blocks chloroplast development. The effect of light is not exerted through induced glycolate formation and appears instead to be photomorphogenetic in character.In senescing leaves excised from the plants decreases in activity of glycolate oxidase, and hydroxypyruvate reductase follow with some delay the decrease in chlorophyll content. The activity of catalase, however, is maintained at high levels, especially when the detached shoots are kept in light.

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... In homogenized plant samples, total chlorophyll a and b, as well as carotenoids, were measured after extraction in acetone [36]. The photorespiration-related essential enzymes (GO, glycolate oxidase and HPR, hydroxy pyruvate reductase) activities were assessed (Feierabend and Beevers [37]). Moreover, the ratio of glycine/serine ratio was known as an indicator of photorespiration [38]. ...
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Accumulation of heavy metals in soil is becoming an increasingly serious eco-environmental problem. Thus, investigating how plants mitigate heavy metal toxicity is necessary to reduce the associated risks. Here, we aimed to assess the bioremediation and stress defense strategies of tolerant plant species grown under complex heavy metals contamination. To this end, a field study was conducted on the vegetation cover of sites with different soil pollution levels. Forty-two plant species that belong to 38 genera and 21 families were identified. The pollution had a significant impact on plant richness in the polluted sites. Out of several screened plants, Amaranthus retroflexus L. was selected because of its high relative density (16.7) and a high frequency (100%) in the most polluted sites. The selected species showed a high biological concentration factor (BCF) and low translocation factor (TF) for Cu, As and Ni. To control the heavy metal-induced oxidative damage, A. retroflexus invested in detoxification (metallothionein and phytochelatins, glutathione and glutathione-S-transferase (GST). At the organ level, oxidase damage (H2O2, lipid and protein peroxidation) was observed, particularly in the roots. To mitigate heavy metal oxidative stress, antioxidant mechanisms (e.g., tocopherols, glutathione, peroxidases, catalase, peroxide dismutase and ASC-GSH cycle) were upregulated, mainly in the roots. Overall, our results suggested the potentiality of A. retroflexus as a promising bioremediatory and stress-tolerant plant at the same time; moreover, defense and detoxification mechanisms were uncovered.
... All enzyme assays were conducted at 25°C using a Tecan Infinate 200 plate reader (Tecan Deutschland) in combination with the Magellan data analysis software (Tecan Austria). The oxidase activity assays were conducted according to a modified protocol (Feierabend and Beevers, 1972). The standard reaction mixture contained 100 mM Tris-HCl, pH 7.5, 5 mM substrate, 0.5 mM EDTA, 0.01 mM FMN, 5 mM MgCl 2 , and 4 mM phenylhydrazine in a final volume of 0.2 mL. ...
Article
In roots of Arabidopsis thaliana L-lactate is generated by reduction of pyruvate via L-lactate dehydrogenase (L-LDH), but this enzyme does not efficiently catalyse the reverse reaction. Here, we identify the A. thaliana glycolate oxidase (GOX) paralogs GOX1, GOX2 and GOX3 as putative L-lactate metabolizing enzymes based on their homology to CYB2, the L-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses L-lactate with a similar efficiency as glycolate; in contrast the photorespiratory isoforms, GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than L-lactate. The key factor making GOX3 more efficient with L-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize L-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-of-function and overexpressor plants indicate that L-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on L-lactate as sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicting that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss-of-function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes L-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of L-lactate after its formation under normoxia. Copyright © 2015, Plant Physiology.
... The change in NADH extinction coefficient of 6.2 mM −1 cm −1 at 340 nm was used to follow the enzymatic activity. Glycolate oxidase activity (GO) was measured according to Feierabend and Beevers [46] by following the formation of glyoxylate complex with phenylhydrazine (ε 324 = 17 mM −1 cm −1 ). ...
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Vanadium (V) can be beneficial or toxic to plant growth and the interaction between arbuscular mycorrhizal fungi (AMF) and V stress was rarely investigated at physiological and biochemical levels of plant groups (C3 and C4) and organs (roots and shoots). We tested the potential of AMF to alleviate the negative effects of V (350 mg V/Kg soil) on shoots and roots of rye and sorghum. Relative to sorghum (C4), rye (C3) showed higher levels of V and lower levels of key elements under V stress conditions. V inhibited growth, photosynthesis, and induced photorespiration (increased HDR & GO activities) and oxidative damage in both plants. AMF colonization reduced V stress by differently mitigating the oxidative stress in rye and sorghum. This mitigation was accompanied with increases in acid and alkaline phosphatase activities in plant roots and increased organic acids and polyphenols exudation into the soil, thus reduced V accumulation (29% and 58% in rye and sorghum shoot, respectively) and improved absorption of mineral nutrients including Ca, Mg and P. AMF colonization improved photosynthesis and increased the sugar accumulation and metabolism. Sugars also acted as a supplier of C skeletons for producing of antioxidants metabolite such as ascorbate. At the antioxidant level, rye was more responsive to the mitigating impact of AMF. Higher antioxidants and detoxification defence system (MTC, GST, phenolics, tocopherols and activities of CAT, SOD and POX) was recorded for rye, while sorghum (C4) improved its GR activity. The C3/C4-specificity was supported by principal component analysis. Together, this study provided both fundamental and applied insights into practical strategies to mitigate the phytotoxicity hazards of V in C3 and C4 grasses. Moreover, our results emphasize the importance of AMF as an environment-friendly factor to alleviate stress effects on plants and to improve growth and yield of unstressed plants.
... The activities of two key enzymes in the photorespiratory pathway, Glycolate oxidase (GO) and hydroxypyruvate reductase (HPR), were assessed. GO activity was assessed by following up the protocol of Feierabend and Beevers (Feierabend and Beevers, 1972) and that for HPR was measured according to the method described by Schwitzguebel and Siegenthaler (Schwitzguebel and Siegenthaler, 1984). Furthermore, a Waters Acquity UPLC-tqd system was employed to determine the glycine (GLY) and serine (SER) contents . ...
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So far, the phytotoxic hazards of nano-sized mercuric oxide (HgO-NPs) are not investigated. Herein, the phytotoxicity of fully characterized HgO-NPs (100 mg/kg soil), prepared by coprecipitation method, on maize grown under ambient (aCO2, 410 ppm) and elevated CO2 (eCO2, 620 ppm) was investigated. Regardless of CO2 concentration, HgO-NPs treatment increased Hg levels in maize organs. HgO-NPs induced severe oxidative stress in aCO2 grown plants as indicated by reduced growth and photosynthesis and accumulation of reactive oxygen species (ROS), through photorespiration and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities, and lipid and protein oxidation products. Although HgO-NPs increased molecular (polyphenols, flavonoids, tocopherols) and enzymatic (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione peroxidase) antioxidants in shoots of aCO2 plants, but this failed to fight the eruption of increased ROS. On contrary, eCO2 treatment mitigated the HgO-NPs impact by promoting photosynthesis and reducing the Hg-induced ROS production. Moreover, eCO2 promoted ROS detoxification via molecular antioxidants overproduction, enhanced superoxide dismutase, catalase and peroxidases activities, and modulation of reduced ascorbate/oxidized ascorbate and reduced glutathione/oxidized glutathione homeostasis. The combined HgO-NPs + eCO2 treatment also enhanced the glutathione-S-transferase activity. This study suggests that HgO-NPs cause severe phytotoxic hazards and this effect will be less detrimental under future CO2 climate.
... The activity of glycolate oxidase (GO) was estimated through determining the formation of glyoxylate phenylhydrazone by the increase in absorbance at 324 nm using a microplate reader. 34 Moreover, the hydroxy-pyruvate reductase (HPR) activities were measured according to the method outlined in ref. 35. On the other hand, the glycine/serine ratio was calculated frequently as an index to estimate photorespiration; 36 glycine and serine were quantified using a Waters Acquity UPLC-tqd system. ...
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Infestation by parasitic weeds is one of the most important environmental challenges threatening cropping systems worldwide. Among these, branched broomrape (Orobanche ramosa), a root holoparasitic weed, detrimentally affects many crops especially tomato (Lycopersicon esculentum) and causes severe crop losses. The positive role of silicon nanoparticles (SiNPs) on growth and yield of plants grown under stressful conditions has been reported. However, no study had investigated the impact of SiNPs on plant-weed interaction. In this study, we conducted a green-house experiment to assess the physiological implications of SiNPs on tomato under the Orobanche challenge. Orobanche infection alone markedly inhibited tomato growth and photosynthesis (P < 0.0001) and induced oxidative damage via increased photorespiration (P<0.0001) and NADPH oxidase activities (P<0.01). Interestingly, SiNPs significantly reduced the infection severity by reducing both the number and biomass of Orobanche tubercles (13 and 31% decrease, respectively). Moreover, SiNPs dramatically ameliorated the physiological and biochemical disorders imposed by Orobanche in tomato. Consistently, SiNPs strengthened the cell wall of host roots by upregulating lignin biosynthesis that acts as a physical barrier against tubercle haustorial penetration. Moreover, SiNPs caused a noticeable decrease in ROS production and improved both enzymatic and non-enzymatic detoxification systems, the thing that was more pronounced in roots than in shoots of infected tomato seedlings. Such organ-specific responses were confirmed by cluster analysis. Overall, this study suggests that tomato plants treated with SiNPs will be more tolerant to Orobanche infection through enhanced structural and metabolic responses.
... Glycolate oxidase (GOX) activity was determined by the formation of a glyoxylate complex with phenylhydrazine (ε324=17 mM -1 cm -1 ; Feierabend and Beevers, 1972). Hydroxypyruvate reductase (HPR) activity was determined according to Schwitzguebel and Siegenthaler (1984) as the oxidation of NADH that was followed at 340 nm upon hydroxypyruvate addition. ...
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Crop yield stability requires an attenuation of the reduction of yield losses caused by environmental stresses such as drought. Using a combination of metabolomics and high-throughput colorimetric assays, we analysed central metabolism and oxidative stress status in the flag leaf of 292 indica rice (Oryza sativa) accessions. Plants were grown in the field and were, at the reproductive stage, exposed to either well-watered or drought conditions to identify the metabolic processes associated with drought-induced grain yield loss. Photorespiration, protein degradation and nitrogen recycling were the main processes involved in the drought-induced leaf metabolic reprogramming. Molecular markers of drought tolerance and sensitivity in terms of grain yield were identified using a multivariate model based on the values of the metabolites and enzyme activities across the population. The model highlights the central role of the ascorbate-glutathione cycle, particularly dehydroascorbate reductase, in minimizing drought-induced grain yield loss. In contrast, malondialdehyde was an accurate biomarker for grain yield loss, suggesting that drought-induced lipid peroxidation is the major constraint under these conditions. These findings highlight new breeding targets for improved rice grain yield stability under drought.
... The supernatant wasdecanted and immediately used for the enzyme assay. GO was assayed as described by Feierabend and Beevers (1972) with modifications. A volume of assay mixture containing 50 mM Tris−HCl buffer (pH 7.8), 0.009% Triton X-100, 3.3 mM phenylhydrazine HCl (pH 6.8), 50 μL of plant extract, and 5 mM glycolic acid (neutralized to pH 7 withKOH) was used to start the reaction. ...
Article
Assimilatory pathways of S and N are well coordinated, and addition of S increases the biomass and yield in plantsand improves efficiency in the use of N (NUE). However, no available studies have attempted to explain the effects of NaSH application on crop yield and N metabolism. This study investigates the effects of different doses of hydrogen sulfide as NaSH (0, 0.5, 1,2.5 and 5 mM) in the formation and assimilation of NH 4 + in plants of Brassica oleracea L. 'Bronco'. According to our results, treatments of 0.5 and 1 mMNaSH increased biomass while decreasing the NO 3 − concentration. In the treatment 0.5 mM of NaSH, NH 4 + accumulation diminished with the stimulation of the GS activity, resulting in a greater content in certain amino acids (AAs); this also boosted the soluble-protein content, which could be related to the greater biomass found in this treatment. However, 2.5 and 5 mM of NaSH induced the formation and accumulation of NH 4 + by further reducing this as well as photorespiration. This excessive accumulation of NH 4 + may be responsible for the lower biomass in these treatments (2.5 and 5 mM of NaSH).We conclude that excessive accumulation of NH 4 + may be responsible for the decline in biomass in the treatments 2.5 and 5 mM of NaSH. Therefore, the application of 0.5 mM of NaSH could be a beneficial strategy for improving the processes involved in the N assimilation, accompanied by enhanced crop biomass.
... Stomatal conductance (gs, mol CO 2 m-m -2 s -1 ) was measured on the abaxial side of Glycolate oxidase (GO) and hydroxypyruvate reductase (HPR) activities were measured 165 according to (Feierabend and Beevers, 1972;Schwitzguebel and Siegenthaler, 1984), 166 respectively. Moreover, to calculate glycine/serine ratio, frequently used as an index to estimate 167 photorespiration (Kebeish et al., 2007), glycine and serine were quantified using a Waters 168 Acquity UPLC-tqd system (Al Jaouni et al., 2018). ...
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Due to industrialization and expansion of nanotechnology, ecosystem contamination by nanoparticles is likely. Overall, nanoparticles accumulate in environmental matrices and induce phytotoxicity, however future climate (elevated CO2 (eCO2)) may affect the distribution of nanoparticles in ecosystems and alter their impact on plants. In the current study, nickel oxide nanoparticles (NiO-NPs) with an average diameter of 54 nm were synthesized using Triton X-100 and characterized by scanning electron microscopy (SEM), UV-VIS spectroscopy and Fourier transform infrared spectroscopy (FTIR). We have investigated the impact of NiO-NPs at a concentration of 120 mg kg−1 soil, selected based on the results of a preliminary experiment, on accumulation of Ni ions in wheat (Triticum aestivum L.) and how that could influence plant growth, photosynthesis and redox homeostasis under two CO2 scenarios, ambient (aCO2, 400 ppm) and eCO2 (620 ppm). NiO-NPs alone reduced whole plant growth, inhibited photosynthesis and increased the levels of antioxidants. However, improved defense system was not enough to lessen photorespiration induced H2O2 accumulation and oxidative damage (lipid and protein oxidation). Interestingly, eCO2 significantly mitigated the phytotoxicity of NiO-NPs. Although, eCO2 did not affect Ni accumulation and translocation in wheat, it promoted photosynthesis and inhibited photorespiration, resulting in reduced ROS production. Moreover, it further improved the antioxidant defense system and maintained ASC/DHA and GSH/GSSG redox balances. Organ specific responses to NiO-NPs and/or eCO2 were indicated and confirmed by cluster analysis. Overall, we suggest that wheat plants will be more tolerant to NiO-NPs stress under future climate CO2.
... All activity measurements were scaled down for semi-high throughput measurement using a micro-plate reader (Synergy Mx, Biotech Instruments Inc., Winooski, VT, United States). The photorespiration enzyme glycolate oxidase (GO, EC1.1.3.1) was determined according to (Feierabend and Beevers, 1972). GO was measured by the formation of a glyoxylate complex with phenylhydrazine (ε324 = 17 mM −1 cm −1 ). ...
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... Photorespiration enzymes, glycolate oxidase (GO) and hydroxypyruvate reductase (HPR), were determined according to Feierabend and Beevers [58] and Schwitzguebel and Siegenthaler [59] respectively. GO was measured by the formation of a glyoxylate complex with phenylhydrazine (ε 324 = 17 mM −1 cm −1 ), and HPR activity was measured as NADH oxidation (ε 340 = 6.2 mM −1 cm −1 ). ...
... In homogenized shoots, the amounts of chlorophyll a and b, as well as carotenoids, were measured in acetone [33]. The photorespiration-related essential enzymes including GO (Glycolate oxidase) and HPR (hydroxy pyruvate reductase) activities were assessed according to Feierabend and Beevers [34] and Schwitzguebel and Siegenthaler 1984, respectively. Moreover, the glycine/serine ratio known as an indicator of photorespiration) [35]. ...
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Because sewage sludge is contaminated with heavy metals, its disposal in the soil may pose risks to the ecosystem. Thus, heavy metal remediation is necessary to reduce the associated risks. The goal of this research is to introduce a heavy metal resistant species and to assess its phytoremediation, oxidative damage markers and stress tolerance mechanisms. To this end, field research was done to compare the vegetation of polluted sites to that of a healthy site. We found 42 plant species identified in the study, Sesuvium portulacastrum L. was chosen because of its high relative density (10.3) and maximum frequency (100 percent) in the most contaminated areas. In particular, S. portulacastrum plants were characterized by strong Cu, Ni, and As uptake. At the organ level, to control growth reduction and oxidase damage, particularly in roots, increased detoxification (e.g., metallothionein, phytochelatins) and antioxidants mechanisms (e.g., tocopherols, glutathione, peroxidases). On the other hand, flavonoids content and the activity of glutathione-S transferase, glutathione reductase and dehydroascorbate reductase were increased manly in the shoots. These biochemical markers can be applied to select tolerance plant species grown under complex heavy metal contamination. Our findings also introduced S. portulacastrum to reduce soil contamination0associated risks, making the land resource available for agricultural production.
... Isocitrate lyase and malate synthase activities were assayed by the methods of Dixon & Kornberg (1959), glycollate oxidase activity was assayed as described by Feierabend & Beevers (1972), but with GSSG omitted, catalase activity as described by Luck (1965), fumarase activity as described by Racker (1950) and NADP+-linked isocitrate dehydrogenase activity by the procedure of Kornberg & Pricer (1951). Attempts to demonstrate an NAD+-linked isocitrate dehydrogenase activity were unsuccessful. ...
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The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10(-6) mol g(-1) f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10(-3) mol g(-1) f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.
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Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.
Article
1. In etiolated wheat (Triticum aestivum L.) leaves, the development of the microbody enzymes catalase, hydroxypyruvate reductase, and glycolate oxidase was specifically stimulated by short treatments of the seedlings with red light, although the increases were less than observed after treatment with continuous white light. A comparison of the effects of short red and far-red exposures indicated the involvement of phytochrome. 2. Continuous far-red light treatments also enhanced the development of microbody enzymes. Catalase activity continued to increase at a high rate even after return from a prolonged far-red illumination to darkness, while the increase in the activities of glycolate oxidase and hydroxypyruvate reductase fell to the dark rates when the tissue was removed from the light. However, even at higher intensities of continuous far-red light the microbody enzymes reached only considerably lower activities than in white light. During continuous irradiation of equal quantum flux, the microbody enzymes reached higher activities in red than in far-red light, but the highest activities were observed in blue light, which had similar effects as white light. The quantitative difference between the effects of prolonged red or blue light depended also on the seed material and growing conditions. In the presence of the herbicide 3-amino-1,2,4-triazole the increase of glycolate-oxidase activity was reduced in red light but was affected much less, if at all, in blue light. 3. Continuous irradiations with all three light qualities used (red, far-red, blue) influenced the properties of the microbody particles to form a distinct band sharply confined close to an equilibrium density of 1.25 g cm(-3) on sucrose gradients which was not observed in preparations from plant material raised in complete darkness. In preparations from all light-grown plants a special peak in the activity profile of malate dehydrogenase was found in the microbody fraction while it was lacking on gradients from dark-grown leaves. The heights of the activities of malate dehydrogenase as well as of the other enzymes found in the microbody fractions from plants grown in either far-red, red, or blue light differed in the same way as did the activities from total leaf homogenates. 4. Glycolate oxidation by segments of intact leaf tissue was higher with tissue from light- than from dark-grown plants, but after light treatments of different spectral quality its magnitude did not correspond to the extractable activities of glycolate oxidase.
Article
Ultrastructural and biochemical approaches were used to investigate the mode of action of tagetitoxin, a nonhost-specific phytotoxin produced by Pseudomonas syringae pv. tagetis (Hellmers) Young, Dye and Wilkie, which causes chlorosis in developing - but not mature - leaves. Tagetitoxin has no effect on the growth rate or morphology of developing leaves of wheat (Triticum aestivum L.) seedlings. Its cytological effects are limited to plastid aberrations; in both light-and dark-grown leaves treated with toxin, internal plastid membranes fail to develop normally and plastid ribosomes are absent, whereas mitochondrial and cytoplasmic ribosomes are unaffected. The activity of a plastid stromal enzyme, ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), which is co-coded by nuclear and chloroplast genes, is markedly lower in extracts of both light-and dark-grown toxin-treated leaves, whereas the activity of another stromal enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-G-3P-DH, EC 1.2.1.13), which is coded only by the nuclear genome, is significantly lower in extracts of light-grown, but not of dark-grown, treated leaves. The mitochondrial enzymes fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) are unaffected by toxin in dark-grown leaves, but fumarase activity is reduced in light-grown ones. Four peroxisomal enzyme activities are lowered by toxin treatment in both light- and dark-grown leaves. Light- and dark-grown, toxintreated leaves contain about 50% and 75%, respectively, of the total protein of untreated leaves. There are threefold and twofold increases in free amino acids in light-grown and dark-grown treated leaves, respectively. In general, the effects of tagetitoxin are more extensive and exaggerated in light-grown than in dark-grown leaves. We conclude that tagetitoxin interferes primarily with a light-independent aspect of chloroplast-specific metabolism which is important in plastid biogenesis.
Article
Microbody transition during the greening of watermelon cotyledons (Citrullus vulgaris Schrad.) was studied by double immunocytochemical labeling of the glyoxysomal marker enzyme isocitrate lyase and the peroxisomal marker enzyme hydroxypyruvate reductase. In order to analyze the immunocytochemistry, developmental stages representing the glyoxysomal, microbodytransition and peroxisomal stages were chosen, taking into account the time course of enzyme activity and the amounts of the respective antigens. It was shown that during microbody transition, between 83 and 91% of all the tested microbodies contained isocitrate lyase as well as hydroxypyruvate reductase, which was significantly higher than in the glyoxysomal and peroxisomal stages of development. Comprehensive controls precluded labeling artifacts. Our results support the one-population hypothesis first proposed by Trelease et al. (1971, Plant Physiol. 48, 461-465).
Article
Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.
Article
A new procedure was used to purify the peroxisomal matrix enzyme hydroxypyruvate reductase (HPR) from green leaves of pumpkin (Cucurbita pepo L.) and spinach (Spinacia oleracea L.). Monospecific antibodies were prepared against this enzyme in rabbits. Immunoprecipitation of HPR from watermelon (Citrullus vulgaris Schrad.) yielded a single protein with a subunit molecular weight of 45 kDa. Immunohistochemical labeling of HPR was found exclusively in watermelon microbodies. Isolated polyadenylated mRNA from light-grown watermelon cotyledons was injected into Xenopus laevis oocytes. The heterologous in-vivo translation product of HPR exhibited the same molecular weight as the immunoprecipitate from watermelon cotyledons, indicating the lack of a cleavable extra sequence. The watermelon HPR translated in oocytes was imported into isolated glyoxysomes from castor bean (Ricinus communis L.) endosperm and remained resistant to proteolysis after the addition of proteinase K. The HPR did not change its apparent molecular weight during sequestration; however, it may have changed its conformation.
Article
β-Oxidation enzymes were detected both in the mitochondria and microbodies of Arum maculatum L. spadices and Brassica napus L. seeds. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose-density-gradient centrifugation, prevents rapid access of acyl-GoA substrates to matrix βoxidation tes. Thus intact mitochondria showed little β-oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of acyl CoAs to matrix sites. Consequently, in ruptured mitochondria, high β-oxidation enzyme activities were measured.
Article
In general, drought depresses nutrient uptake by the root and transport to the shoot due to a restricted transpiration rate, which may contribute to growth limitation under water deprivation. Moreover, water stress may also restrict the ability of plants to reduce and assimilate nitrogen through the inhibition of enzymes implicated in nitrogen metabolism. The assimilation of nitrogen has marked effects on plant productivity, biomass, and crop yield, and nitrogen deficiency leads to a decrease in structural components. Plants produce significant quantities of NH4+ through the reduction of NO3− and photorespiration, which must be rapidly assimilated into nontoxic organic nitrogen compounds. The aim of the present work was to determine the response of reciprocal grafts made between one tomato tolerant cultivar (Lycopersicon esculentum), Zarina, and a more sensitive cultivar, Josefina, to nitrogen reduction and ammonium assimilation under water stress conditions. Our results show that when cv. Zarina (tolerant cultivar) was used as rootstock grafted with cv. Josefina (ZarxJos), these plants showed an improved N uptake and NO3− assimilation, triggering a favorable physiological and growth response to water stress. On the other hand, when Zarina was used as the scion (JosxZar), these grafted plants showed an increase in the photorespiration cycle, which may generate amino acids and proteins and could explain their better growth under stress conditions. In conclusion, grafting improves N uptake or photorespiration, and increases leaf NO3− photoassimilation in water stress experiments in tomato plants.
Article
1. In developing rye (Secale cereale L.) leaves the formation of plastidic ribosomes was selectively prevented in light as well as in darkness, when the seedlings were grown at an elevated temperature of 32° instead of 22° where normal development ocurred. Plastid ribosome deficient parts of lightgrown leaves were chlorotic at 32°. - 2. At both temperatures the leaves contained under all conditions (light or dark, on H2O or nutrient solution) equal or very similar amounts of total amino nitrogen. In light, the contents of total protein and dry weight were lower at 32° than at 22°, especially when the plants were grown on nutrient solution. - 3. Mitochondrial marker enzymes had normal or even higher activities in 32°-grown leaves. Respiration rates were similar for segments of leaves grown on water in light either at 32° or at 22° but by 20-30% lower for 32°-grown plants when they had been raised in darkness or on nutrient solution. In contrast to 22°-grown tissue, respiration of 32°-grown leaf segments was rather insensitive to KCN. Comparative inhibitor studies indicated the presence of both the cyanide-sensitive and the cyanide-insensitive pathway of respiration in 32°-grown leaves. - 4. Leaf microbody marker enzymes were present in leaves grown at 32°. From chlorotic parts of 32°-light-grown leaves a typical microbody fraction was isolated on sucrose densitygradients. - 5. Leaves of seedlings grown at 32° contained only very low levels of ribulosediphosphate carboxylase activity and of fraction I protein. Photosynthetic (14)CO2-fixation of such leaves was only a few per cent of that observed in normal leaves, and no photosynthetic oxygen evolution was observed in chlorotic leaf segments. However, ten other soluble enzymes which are exclusively or partially localized in chloroplasts reached high activities under all conditions at 32° (Table 4). - 6. From chlorotic parts of 32°-light-grown leaves as well as from etiolated 32°-grown leaves a fraction of intact plastids was isolated and purified by sucrose gradient centrifugation which contained several soluble chloroplast enzymes. From the results we conclude that cytoplasmic protein synthesis must contribute a functional chloroplast envelope including the mechanism for the recognition and uptake of chloroplast proteins which are synthesized on cytoplasmic ribosomes.
Article
The plant growth substance jasmonic acid and its methyl ester (JA-Me) induce a set of proteins (jasmonate-induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one-dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno-gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP-23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP-37 was detected in vacuoles and in the nucleoplasm; JIP-66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP-100 was found within the stromal fraction of chloroplasts; JIP-70 is present in the peroxisome and the nucleus; JIP-50 and JIP-6 accumulate in vacuoles. The location of JIP-66 and JIP-6 confirms their possible physiological role deduced from molecular analysis of their cDNA.
Article
In contrast to an earlier publication (Drumm et al., Cytobiol. 2, 335, 1970), a definite enhancement by phytochrome of the catalase level in mustard (Sinapis alba L.) cotyledons can be demonstrated. This response can be obtained either with continuous far-red light or with short red pulses, the effect of which is reversible by short far-red pulses. From the comparison of the time courses of catalase activity with the time courses of glyoxysomal (isocitrate lyase) and peroxisomal (glycolate oxidase, glyoxylate reductase) marker enzymes in dark grown and far-red irradiated cotyledons, there appears to be a close relationship between the catalase present in darkness and glyoxysomes and between the phytochrome-stimulated portion of total catalase and peroxisomes, respectively. The isoenzyme pattern of catalase shows 3 strong and several weaker bands in dark grown cotyledons. Irradiation with white or far-red light leads to a more complex pattern with at least 12 detectable bands. The isoenzymes increased by light supplement rather than replace the isoenzymes present in darkness. This is true also in cotyledons and true leaves of white light grown plants which do not possess glyoxysomes. In the hypocotyl of the seedling, catalase formation is depressed by far-red light and no change in the isoenzyme pattern is observed. It is concluded that the development of peroxisomes in the cotyledons is specifically controlled by phytochrome and that this subcellular differentiation also involves the control of catalase, a marker enzyme for both glyoxysomes and peroxisomes. The implications of these results with respect to the developmental origin of peroxisomes in cotyledons of fat-storing, potentially photosynthetically active cotyledons is discussed.
Chapter
All the cells of a wheat leaf are derived from meristematic cell division at the leaf base. Consequently, cell maturity increases towards the leaf tip as does the size, number and photosynthetic activity of the chloroplasts (1–3). Rapid changes in plastid morphology and biochemistry within light-grown wheat leaves have received much attention whereas the extent to which other leaf cell organelles collaborate, during maturation of the mesophyll cell, has been largely overlooked.
Article
Industrial activities have led to a gradual and global increase in soil aluminum (Al) and atmospheric CO2 concentrations. Al bioavailability strongly depends on the soil pH, which in turn is affected by atmospheric CO2 levels. In spite of the concurrent impact which Al and elevated CO2 (eCO2) could have on plants, their interaction and how it affects the growth of economically important crop species has not been investigated. Here, we have investigated the synchronous impact of soil Al and eCO2 exposure on key C3 (wheat, oat) and C4 (maize, sorghum) crops, at the physiological and biochemical level. Compared to C3 plants, C4 plants accumulated less Al by stimulating soil Al retention through exudation of root organic acids. Consequently, C4 plants maintained photosynthetic performance and anti-oxidative capacity. However, under eCO2 the differential responses of C3 and C4 crops to Al exposure were reduced. Elevated CO2 decreased Al accumulation and oxidative damage in all cereals, and ameliorated C3 plant growth specifically. This was reflected on the biochemical level, where eCO2 inhibited ROS production and restored RuBisCo activity in C3 crops only. Overall, C4 cereals appear more tolerant to soil Al exposure under current ambient CO2 (aCO2) levels, C3 crops could attain comparable tolerance under future eCO2 conditions.
Article
Ultrastructural evidence for structures resembling microbodies is presented for the fungus Achlya ambisexualis Raper. These structures are DAB positive and thus presumably contain the enzyme catalase. Activities from mycelial homogenates for. the following enzymes are given: catalase, glycolate oxidase, uricase, isocitrate lyase, malate dehydrogenase, citrate synthetase, malate synthetase and glutamate: oxaloacetate transaminase. These results suggest that Achlya contains microbodies and that they may be of the glyoxysome type. The specific activity of catalase increases substantially following initiation of antheridial hyphae by the hormone antheridiol.
Chapter
The determination of enzyme activities in organ or organellar extracts is an important means of investigating metabolic networks and allows testing the success of enzyme-targeted genetic engineering. It also delivers information on intrinsic enzyme parameters such as kinetic properties or impact of effector molecules. This chapter provides protocols on how to assess activities of the enzymes of the core photorespiratory pathway, from 2-phosphoglycolate phosphatase to glycerate 3-kinase.
Article
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Under normal and stress conditions plant growth require a complex interplay between phytohormones and reactive oxygen species (ROS). However, details of the nature of this crosstalk remain elusive. Here, we demonstrate that PINOID (PID), a serine threonine kinase of the AGC kinase family, perturbs auxin homeostasis, which in turn modulates rosette growth and induces stress responses in Arabidopsis plants. Arabidopsis mutants and transgenic plants with altered PID expression were used to study the effect on auxin levels and stress-related responses. In the leaves of plants with ectopic PID expression an accumulation of auxin, oxidative burst and disruption of hormonal balance was apparent. Furthermore, PID overexpression led to the accumulation of antioxidant metabolites, while pid knockout mutants showed only moderate changes in stress-related metabolites. These physiological changes in the plants overexpressing PID modulated their response toward external drought and osmotic stress treatments when compared to the wild type. Based on the morphological, transcriptome, and metabolite results, we propose that perturbations in the auxin hormone levels caused by PID overexpression, along with other hormones and ROS downstream, cause antioxidant accumulation and modify growth and stress responses in Arabidopsis. Our data provide further proof for a strong correlation between auxin and stress biology.
Article
Changes of activity of nitrate reductase, glycerate dehydrogenase, triosephosphate isomerase, malate dehydrogenase, isocitrate dehydrogenase and shikimate dehydrogenase were investigated in different parts of primary leaves of wheat and barley during leaf development. Nitrate reductase and glycerate dehydrogenase show marked changes in activities. The maximum of activity shifts basipetally during leaf development. This is due to the different physiological ages of cells in different parts of the leaves. Nitrate reductase exhibits its maximum of activity during the phase of growing and a suppression occurs before full activity of the photosynthetic apparatus appears.
Chapter
Peroxisomes and glyoxysomes, often referred to under the general heading of “microbodies”, represent analogous cell organelles which compartmentalize the H2O2-producing sections of metabolic pathways. Microbodies are eukaryotic and thus are not exclusively plant organelles as plastids appear to be. However, in plants these organelles are involved in some very important metabolic pathways which are unique to the plant kingdom. In the fat-storing tissue of seedlings glyoxysomes are involved in the degradation of stored lipids and are thus essential for the mobilization of carbohydrates during the first days of germination. The glyoxysomal function of microbodies has also been described in some fungi and animals. In green leaves the peroxisomes house part of the photorespiration pathway which is unique to plants. Leaf peroxisomes play an important role in minimizing the photodestruction of the chlorophyll in C3-plants. Basically identical organelles are found in C4- and CAM-plants (Gross and Beevers, 1989; Herbert et al., 1978). Another important and plant-specific function of microbodies is their involvement in nitrate metabolism within the root nodules of fabaceous species.
Chapter
Since May 1987, we have set up 8 open-top chambers for the study of the physiology of trees under controlled stress conditions. Since 1989, two years after acclimatization, the atmosphere of the enclosures has been progressively enriched in CO2. The CO2 content in the air was raised to 480 and 570 ppmV respectively in two of the open-top chambers. In the four other chambers, the atmosphere was not enriched with gas.
Chapter
Euglena gracilis KLEBS var. bacillaris PRINGSHEIM offers many inducements to students of organelle development and inheritance. SEYMOUR HUTNER, LUIGI PROVASOLI and their numerous co-workers at Haskins Laboratories persuaded the organism to grow luxuriently in axenic culture on a variety of completely defined media over the extraordinarily wide pH range of three to eight. They also showed that this strain of Euglena could be made to lose visible plastids and pigmentation reversibly by growth in the dark and irreversibly through growth on streptomycin (PROVASOLI et al. 1948). CRAMER & MYERS (1952) studied its photosynthetic properties while PRINGSHEIM & PRINGSHEIM (1952) who had originally isolated this strain, also showed that growth above 32 °C would bring about irreversible loss of plastids. Out of these observations has grown a body of knowledge and techniques for exploiting these useful attributes.
Article
We have examined the activity and regulation of the glycolate oxidizing enzyme in ten different greening stages of the Chlorella mutant Chlorella fusca C-1.1.10.10 The mutant has lost the ability to form chlorophyll in the dark. The dark grown cells exposed to light need after a short lag phase about 12 hs for normal greening. CO2-fixation starts after 2 h of illumination. In the first 2 h of illumination we find a phenomenon in the induction of the enzyme activity. In the first hour the activity increases to a maximum and then decreases in the second hour to a minimum. After a recovery time the activity increases proportionally to the time of illumination and finally reaches a value about 8 times higher than that found in the normal Chlorella strain. These results are discussed with respect to a dual mechanism in the regulation of the enzyme activity. At the beginning the glycolate oxidation may be regulated by phytochrome and then by a substrate induction.
Chapter
Light affects the development of plastids in many different ways: Via phototransformation of protochlorophyllide into chlorophyllide a, via the phytochrome system, and via the two photosystems of photosynthesis. All these pigment systems respond to both blue and red light. It is, however, quite clear that in many cases blue light has special effects which cannot be duplicated by red light. This holds both for various algae (as will be shown by several other contributions in this book) and for higher plants. The present treatise will deal with effects induced specifically by blue light (in this term I include violet and near-ultraviolet radiation) in higher plants (among which I include ferns).
Chapter
The early research on peroxisomes in plants was centred mainly on three groups, my own at Purdue (later at University of California, Santa Cruz) and those of Tolbert at Michigan State University and Newcomb at Wisconsin. Some of the collaborators from these centres subsequently established their own research laboratories and, as interest in the field expanded, others independently joined the international association. Several authors in the present volume trace their lineage in peroxisomal research either directly or indirectly to these three groups, but progress has been such that the questions they now address are well beyond the vision of their ancestors.
Chapter
Growth, differentiation and the integration of these processes ( = morphogenesis) in the multicellular plant is profoundly influenced by light acting through photomorphogenetic sensor and effector molecules, from which phytochrome is the one most widely known. It is obvious that a similar situation exists at the next lower level in the hierarchy of complexity, i.e., at the level of the cell which functions as an integrated system of numerous subcellular compartments. A major reason for studying the functional and structural changes within the cell during photomorphogenesis is the hope that the regulatory processes governing the coordinate development of subcellular compartments may, at the present state of experimental arts, be more open for a successful attack than those directing photomorphogenesis of the whole plant. (In its strict sense this problem has been hardly addressed up till now). Indeed the last few years have seen the advent of powerful new techniques for the in vitro investigation of basic cellular processes such as DNA transcription and RNA translation by the various genetic systems, or the specific transport of functional proteins from one cell compartment to another. These techniques are now being explored in the research on intracellular photomorphogenesis. It appears appropriate, therefore, to devote this chapter mainly to a critical review of the recent molecular approaches in this field.
Chapter
Photosynthesis depends on light. Looking deeper into this obvious dependence opens some important aspects of the overall photosynthetic process. In Fig. 13 the efficiency of photosynthetic CO2-fixation in different regions of the visible spectrum of sunlight is compared with the absorption spectra of intact cells and of pure chlorophyll a and b. It is obvious that not only chlorophyll but also the absorption by other pigments contributes to the overall CO2-fixation. In the chloroplast grana and stroma lamellae chlorophylls a and b are integrated with “accessory pigments” into the pigment systems I and II (p. 22). These accessory pigments include the yellow, orange, and brownish pigments β-carotene, lutein, violaxanthin, and neoxanthin.
Article
The metabolism of ¹⁴ C-SAN-6706 [4-chloro-5-(dimethylamino)-2-( a,a,a -trifluoro- m -tolyl)-3(2 H )-pyridazinone] and ¹⁴ C-norflurazon [4-chloro-5-(methylamino)-2-( a,a,a -trifluoro- m -tolyl)-3(2 H )-pyridazinone] were studied in cranberry plants ( Vaccinium macrocarpon Ait. ‘Early Black’) for a 36-day period. Both compounds were readily translocated with progressively more label being present in the shoot portion with time. The two compounds are demethylated to the desmethyl analogue SAN-9774 (4-chloro-5-amino-2-( a,a,a -trifluoro- m -tolyl)-3(2 H )-pyridazinone). Berry samples from cuttings contained no detectable radioactivity while samples from 2 year-old cranberry plants contained 20 ppb or less.
Article
The oxidative photosynthetic carbon cycle (or C2 cycle) is the metabolic pathway responsible for photosynthetic oxygen uptake and the light‐dependent production of carbon dioxide that is termed photorespiration. The C2 and reductive C3 cycles coexist, and combined, represent total photosynthetic carbon metabolism. A brief historical review is presented beginning with the early observations of the oxygen inhibition of photosynthesis up to the discovery of the oxygenase activity associated with ribulose 1,5‐bisphosphate carboxylase/oxygenase. The properties and the role of the compartmentalization of the enzymes involved with the pathway and the transport of C2 cycle intermediates are reviewed. The relationship of the C2 cycle to photorespiratory nitrogen metabolism and other associated metabolic pathways and the properties and regulation of the C2 cycle in diverse photosynthetic organisms are discussed.
Article
The developmental changes of marker enzymes of the glyoxysomal function [isocitrate lyase (ICL) and malate synthase (MS)] and the leaf-peroxisomal function [glycolate oxidase (GO) and hydroxypyruvate reductase (HPR)] of the peroxisomes were investigated. White light suppressed the activities of ICL and MS while the activities of GO and HPR were increased. The stimulation of GO and HPR is mediated by continuous far-red light absorbed by phytochrome, whereas photosynthetic pigments are probably responsible for the photoinhibition of ICL and MS. Acetate and glucose had no regulatory effects on the levels of these enzymes. The same result was obtained if CO2-fixation was inhibited by withholding CO2, or if photorespiration was inhibited by withholding O2, in the light. However, Norflurazon (NF), an inhibitor of carotenoid synthesis leading to photooxidative destruction of chloroplasts in the light, prevented the light-mediated increase of GO and HPR while the photoinhibition of ICL and MS was alleviated. The same differential effects could be obtained by inhibiting plastid protein synthesis with Chloramphenicol, Lincomycin, Rifampicin, and a heat treatment (34 °C) destroying plastid ribosomes. Inhibition of cytoplasmic protein synthesis with Cycloheximide completely prevented the formation of all four enzymes. Inhibition of photosynthesis by Dichlorophenyl-dimethylurea (DMCU) in green cotyledons did not specifically influence the light-dependent changes of enzyme levels. NF reduced the light-mediated association of peroxisomes to plastids but had no detectable effect on the fine-structure of peroxisomes, even if located in close contact with photodamaged chloroplasts. From these data it is concluded that (i) the peroxisomal enzyme levels are not controlled by metabolites of fat degradation, photosynthesis, or photorespiration, (ii) a functional photosynthetic apparatus is not required for a normal development of both groups of peroxisomal enzymes in the light, (iii) plastids exert control over the accumulation of these cytoplasmic enzymes which can be disturbed by eliminating plastid protein synthesis. Thus, phytochrome-mediated photomorphogenesis of peroxisomes is regulated by a signal originating in the plastid either independently of, or as a consequence of, phytochrome-mediated plastid development.
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Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from spinach leaves. After grinding leaves in 0.5 M sucrose, the peroxisomes were removed with the broken chloroplast fraction by differential centrifugation. During sucrose density gradient centrifugation, the peroxisomes banded in about 1.9 M sucrose and were separated from mitochondria and chloroplasts. The particles, 0.5 to 1.0 µ in diameter, contained a dense granular stroma surrounded by a single membrane. The leaf peroxisomes contained glycolate oxidase, DPNH-glyoxylate reductase, and catalase. Up to 55% of the activity for these enzymes in spinach leaves have been found in the particulate fractions after the initial centrifugation. The leaf peroxisomes are probably the site of oxygen uptake during photorespiration. No catalase activity was present in chloroplasts after removal of the peroxisomes by density gradient centrifugation. P-Glycolate phosphatase, TPNH-glyoxylate reductase, D-amino acid oxidase, urate oxidase, and peroxidase were not present in leaf peroxisomes.
Article
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A NADH-hydroxypyruvate reductase was located in peroxisomes isolated from spinach leaves. The enzyme was precipitated by between 15 to 23 g of (NH4)2SO4 per 100 ml and had a pH optimum at 6.4. It was also active with NADPH at a pH optimum of 5.1, and the NADH:NADPH ratio of maximum activity was about 13:1. The reductase with either NADH or NADPH was about 4.3-fold more active with hydroxypyruvate than with glyoxylate. Through separation of proteins by electrofocusing, by Sephadex G200 column chromatography, and by starch gel and polyacrylamide gel electrophoresis, it was shown that a single peroxisomal protein catalyzed these reactions. The enzymic activity with NADH was not significantly affected by salts, except for nitrate, which was inhibitory. Activity with NADPH was inhibited by salts. A NADPH-glyoxylate reductase was located in the chloroplasts, which were separated from other particles by isopycnic sucrose density gradient centrifugation. It reduced glyoxylate 16-fold faster than hydroxypyruvate and was highly specific for NADPH. The enzyme was precipitated by between 30 to 45 g of (NH4)2SO4 per 100 ml and had a pH optimum of 6.2. Maximum activity per g of tissue of the chloroplast reductase, as measured with NADPH and glyoxylate, was about 1/20 that of the peroxisomal enzyme measured with NADH and hydroxypyruvate. Peroxisomes may function for disposal of excess reducing equivalents from photosynthesis via a glycolate-glyoxylate shuttle. Excess NADPH formed in the chloroplasts is consumed in the reduction of glyoxylate to glycolate, which is then oxidized in the peroxisome. Glyoxylate may return to the chloroplast to complete the cycle or be further metabolized in the peroxisomes. The peroxisomal reductase functions in this latter process for the reduction of hydroxypyruvate to glycerate.
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Peroxisomes from spinach leaves contain three aminotransferases, a glutamate-glyoxylate, a serine-pyruvate and an aspartate aminotransferase. Five other enzymes previously described in leaf peroxisomes were measured, namely, catalase, glycolate oxidase, NADH-hydroxypyruvate reductase, and NAD-malate dehydrogenase. A NADPisocitrate dehydrogenase was also associated with isolated leaf peroxisomes, but its specific activity was much less than the other enzyme. Most of the NADP-isocitrate dehydrogenase activity was soluble. Serine-pyruvate aminotransferase activity in the particulate fraction was localized entirely in the peroxisomes. Isoenzymes of asparate aminotransferase were found in chloroplasts, mitochondria, and peroxisomes. The activities of the chloroplast and mitochondrial forms of this aminotransferase were greater than the peroxisomal isoenzyme. The two peroxisomal serine-pyruvate and aspartate aminotransferases were separated by disc electrophoresis and isoelectric focusing. The aminotransferases had different substrate specificity. Other enzymes of the tricarboxylic acid cycle, with the exception of an isoenzyme of NAD-malate dehydrogenase, were detected in mitochondria but not in the leaf peroxisomal fractions. Enzymes for the reductive amination of α-keto acids, the interconversion of C4 and C3 acids, hydroxyaspartate dehydratase, and the α and β oxidation systems for fatty acids could not be detected in leaf peroxisomes. Preliminary evidence is presented for a malate synthase in leaf mitochondria.
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Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose. In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial. Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane. Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration. Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.
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Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm(3) and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm(3). During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm(3). However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm(3) and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm(3) was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.
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The cells of higher organisms contain highly differentiated bodies or organelles, each of which is delimited from the surrounding cytoplasm by an outer membrane. Organelles carry out specialized cellular functions; they possess complex and unique internal structures and contain a characteristic complement of enzymes and other constitutents. Clearly, knowledge of the genetic and metabolic regulatory systems involved in the differentiation of these cellular structures is crucial to our comprehension of the biochemistry of growth and differentiation of the cells of higher organisms.
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1. A method was developed for the measurement of glycollate oxidase activity in buffered extracts of white mustard seedlings (Sinapis alba L.). 2. After 36 hours germination in darkness, the activity of the enzyme was followed during the further development of the seedlings kept in darkness or irradiated continuously with red or far-red light. In irradiated seedlings as well as in dark-grown controls, most of the activity was found in the cotyledons. 3. Kinetics of activity induction were studied for both the whole seedlings and the cotyledons. In permanent darkness enzymatic activity increased up to 60 hours after sowing; then a nearly constant activity level was maintained. When the seedlings were exposed to continuous far-red light, an increase of enzymatic activity was observed. This light-mediated increase showed a lag-phase wich was dependent on the age of the seedlings. When irradiation was started 36 hours after sowing, it took at least 12 hours for the differences between light and dark activity to become significant; in seedlings irradiated 60 hours after sowing, the lag-phase -if any- was less than 6 hours. The magnitude of the photoresponse diminished with increasing age of the seedlings. After interruption of irradiation in the phase of vigorous activity induction, the activity kept rising for about 8 hours and resulted in a stable intermediary activity level. Continuous far-red light, started 36 hours after sowing, caused a threefold activity increase in cotyledons after 42 hours and the maximal level of activity was light-independent. An activity induction was also obtained under continuous irradiation with red light. When irradiation was started 36 hours after sowing, a lag-phase of about 6 hours was found. Then the activity rose to a constant level inferior to that in continuous far-red light. With 60 hours old seedlings, no significant differences were observed in the response to either red or far-red light. 4. The involvement of phytochrome in the photoresponse was shown by means of the conventional induction-reversion experiments. 5. The induction pattern of glycollate oxidase in mustard is compared with that of other glyoxysome enzymes and dicussed with respect to the problem of compartment biogenesis. Dissimilarities in the induction patterns of the individual enzymes indicate the absence of a uniform regulatory mechanism for the entire glyoxysome compartment of the cells.
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Single membrane bounded organelles characterized by a physical association with endoplasmic reticulum have been observed in a wide range of cell types and plant species including Gymnosperm, Angiosperm, Pteridophyte, and Thallophyte (algae and fungi) tissues. The morphological similarity between these organelles and animal microbodies suggests that they are cytological homologues. Plant microbodies were observed both with and without dense internal inclusions but unlike animal microbodies could not be shown to contain uricase. Plant microbody membranes are resistant to degenerative influences and remain associated with a small portion of endoplasmic reticulum even in isolated cell fractions.
Article
1. Factors were investigated which control the formation of photosynthetic enzymes during germination. Enzymes of the reductive pentose phosphate cycle like carboxydismutase (EC 4.1.1.39) and NADP-dependent glyceraldehydephosphate dehydrogenase (EC 1.2.1.9.) are formed in the primary leaves of dark-grown rye seedlings. The rate of their synthesis is determined by the level of cytokinins. This rate can be increased by treatment of normal seedlings with kinetin. After application of kinetin to dark-grown seedlings, the investigated enzymes finally reach the same activity as they do in untreated light-grown plants. The formation of these photosynthetic enzymes can be strongly reduced by excision of the roots early in the development, a treatment which is known to lower the supply of cytokinins. A high rate of enzyme formation can be restored by feeding kinetin to rootless seedlings. Neither adenosine nor gibberellic acid have this effect on enzyme formation. 2. Changes in the content of cytokinins preferentially influence the formation of the investigated photosynthetic enzymes. Some cytoplasmic enzymes are not affected by the decrease of the cytokinin level which is achieved by excision of the roots. At the beginning of germination only cytoplasmic enzymes are promoted by application of kinetin, whereas in later stages, after 96 hours of germination, only the formation of photosynthetic enzymes is increased. The formation of photosynthetic and cytoplasmic enzymes seem to differ in their cytokinin requirements. 3. Cytokinins seem to be necessary for the formation of enzymes of the reductive pentose phosphate cycle. However, the cytokinins do not alter the time of appearance of these enzymes. Also the suppressing action which is exerted on the formation of photosynthetic enzymes by low temperature cannot be prevented by the application of kinetin. The action of cytokinins probably does not induce the derepression of the genes, but the level of cytokinins determines the extent of the manifestation of the genes. 4. The formation of the photosynthetic enzymes is also promoted by phytochrome. Phytochrome and cytokinin act as independent factors in a multiplicative system. The rate of synthesis of these enzymes in the dark, which corresponds to the cytokinin level of the seedlings (rootless, normal or treated with kinetin) can be increased by a constant factor via the phytochrome system by continuous irradiation with far-red light. In the case of carboxydismutase this factor is nearly 2. 5. After excision of the roots carboxydismutase and NADP-dependent glyceraldehydephosphate dehydrogenase reach higher activity in red and blue light than in far-red light, under which no chlorophyll is formed. In this case formation of carboxydismutase in red and blue light seems to proceed in close correlation with chlorophyll synthesis.
Article
Morphology and distribution of the relatively less well known organelles of plants have been studied with the electron microscope in tissues fixed in glutaraldehyde and postfixed in osmium tetroxide. An organelle comparable morphologically to the animal microbody and similar to the plant microbody isolated by Mollenhauer et al. (1966) has been encountered in a variety of plant species and tissues, and has been studied particularly in bean and radish roots, oat coleoptiles, and tobacco roots, stems and callus. The organelle has variable shape and is 0.5 to 1.5 μ in the greatest diameter. It has a single bounding membrane, a granular to fibrillar matrix of variable electron density, and an intimate association with one or two cisternae of rough endoplasmic reticulum (ER). Microbodies are easily the most common and generally distributed of the less well characterized organelles of plant cells. It seems very probable that they contain the enzymes characteristic of animal lysosomes (containing hydrolases) or animal microbodies (containing catalase and certain oxidases). Spherosomes are also possible sites of enzyme activity but are not as common or as widely distributed as microbodies. For this reason it appears likely that the particles designated as “plant lysosomes”, “spherosomes”, “peroxisomes”, etc., in some of the cytochemical and biochemical studies on enzyme localization will prove to be microbodies. Variations in the morphology and ER associations of microbodies in tissues of bean and radish are described and discussed. “Crystal-containing bodies” (CCBs) are interpreted as a specialized type of microbody characteristic of metabolically less active cells. Stages in the formation of CCBs from microbodies of typical appearance are illustrated for Avena. The general occurrence of microbodies in meristematic and differentiating cells and their close association with the ER suggest that they may play active roles in cellular metabolism. The alterations in their morphology and numbers that are observed in certain differentiating cells suggest further that the enzyme complements and metabolic roles of microbodies might change during cellular differentiation. If so, microbodies could be the functional equivalent of both microbodies and lysosomes of animal cells.
Article
Chloroplast ribosomes from tobacco leaves show the same stereospecificity of inhibition by chloramphenicol as bacterial ribosomes do. Cytoplasmic ribosomes from the same leaves are unaffected by chloramphenicol. These results remove doubts raised by nonstereospecific effects of chloramphenicol on higher plant cells and support the concept that chloroplasts have evolved from prokaryotes.
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Far red light reversal of red light induced leaf expansion and enzyme changes were investigated in seedlings of Phaseolus vulgaris var. Black Valentine. In etiolated plants growth, anthocyanin accumulation and increases in glyceraldehyde-3-phosphate dehydrogenase and glycolic acid oxidase activities induced by a 10 min red irradiation were stopped by a 7 min far red irradiation given 17, 24, or 48 hr after activation. Etiolated seedlings illuminated for 24 hr with white light and seedlings grown in continuous light remained sensitive to far red reversal. This suggests that the far red sensitive receptor does not decay with time but remains associated with the site of its regulatory functions.
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Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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A spectrophotometric method of measuring the enzymatic formation and disappearance of umaric and cis-aconitic acids is reported.RésuméNous décrivons une methode spectrophotométrique qui permet de mesurer la formation et la disparation enzymatique de l'acide fumarique et de l'acide cis-aconitique.ZusammenfassungEine spektrophotometrische Methode zur Messung der enzymatischen Bildung und Zerstörung von Fumarsäure und cis-Akonitsäure wird beschrieben.
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The literature contains numerous references (e.g., Bonner (1), Van Fleet (18), Weier and Stocking (19)) to the occurrence of catalase, cytochrome oxidase, peroxidase, phosphatase, and polyphenolase activities in higher plants, and an occasional report has appeared dealing with certain of these activities in corn. Thus there have been reports of cytochrome oxidase (4, 9), peroxidase (2), catalase (2, 3, 13), and phosphatase (8) activities in preparations- of various corn tissues. In most cases, however, only one stage of development has been considered. The experiments reported here were undertaken when, in connection with other studies in progress at this laboratory, it became necessary to obtain estimates of the five types of activity in preparations of corn embryos, etiolated shoots, and green seedlings. The aim of these experiments was threefold: to adapt published assay methods for use with preparations of corn tissue, to estimate the activities of preparations from corn at the various stages of development listed above, and to study the fractionation of activities effected by high-speed centrifugation.
Article
Although cycloheximide is extremely inhibitory to protein synthesis in vivo in higher plants, the reported insensitivity of some plant ribosomes suggests that it may not invariably act at the ribosomal level. This suggestion is reinforced by results obtained with red beet storage tissue disks, the respiration of which is stimulated by cycloheximide at 1 microgram per milliliter. Inorganic ion uptake by these disks is inhibited by cycloheximide at 1 microgram per milliliter while the uptake of organic compounds, by comparison, is unaffected. Ion uptake by all nongreen tissues tested is inhibited by cycloheximide, but leaf tissue is unaffected, indicating that the ion absorption mechanism in the leaf may differ fundamentally from that in the root. It is concluded that cycloheximide can affect cellular metabolism other than by inhibiting protein synthesis and that the inhibition of ion uptake may be due to disruption of the energy supply.
Article
The phytochrome controlled increase in total protein in the primary leaf pair of etiolated bean (Phaseolus vulgaris var. Black Valentine) seedlings, which occurs during growth in the dark subsequent to a brief illumination, was investigated. Enzymes from the chloroplasts, the mitochondria, and the soluble cytoplasm all increase in total activity after the illumination.The total protein and the ribulose carboxylase increases are not inhibited by FUdR, an inhibitor of DNA synthesis. Cycloheximide, an inhibitor of protein synthesis, applied at a time when the ribulose carboxylase activity increase has already commenced, blocks further increase. It was concluded that the total protein and the enzyme increases in the leaf are the result of increases in the per cell levels.The initial brief illumination is saturating, but 40 minutes later the seedlings have acquired the ability to respond to a second brief illumination. The rate of increase in ribulose carboxylase activity in seedlings that have been illuminated twice is greater than the rate in seedlings that have been illuminated only once.Far-red light prevents further increase in enzyme activity 48 hours after the initial illumination. There is a lag period interposed between the time of illumination with far-red light and the time at which the seedlings show the greatest effect of far-red light. It was concluded that the phytochrome influence on protein synthesis is not at the terminal steps.
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