Plastid Fraction Research Articles

Subcellular distribution of aluminum associated with differential cell ultra-structure, mineral uptake, and antioxidant enzymes in root of two different Al+3-resistance watermelon cultivars

Crop plants, such as watermelon, suffer from severe Aluminum (Al3+)-toxicity in acidic soils with their primary root elongation being first arrested. However, the significance of apoplastic or symplastic Al3+-toxicity in watermelon root is scarcely reported. In this work, we identified a medium fruit type (ZJ) and a small fruit type (NBT) as Al+3-tolerant and sensitive based on their differential primary root elongation rate respectively, and used them to show the effects of symplastic besides apoplastic Al distribution in the watermelon's root. Although the Al content was higher in the root of NBT than ZJ, Al+3 allocated in their apoplast, vacuole and plastid fractions were not significantly different between the two cultivars. Thus, only a few proportion of Al+3 differentially distributed in the nucleus and mitochondria corresponded to interesting differential morphological and physiological disorders recorded in the root under Al+3-stress. The symplastic amount of Al+3 substantially induced the energy efficient catalase pathway in ZJ, and the energy consuming ascorbate peroxidase pathway in NBT. These findings coincided with obvious starch granule visibility in the root ultra-structure of ZJ than NBT, suggesting a differential energy was used in supporting the root elongation and nutrient uptake for Al+3-tolerance in the two cultivars. This work provides clues that could be further investigated in the identification of genetic components and molecular mechanisms associated with Al+3-tolerance in watermelon.

Purification of Nongreen Plastids (Proplastids and Amyloplasts) from Angiosperms, and Isolation of Their Envelope Membranes.

Plastids, a wide family of plant specific organelles, exist in all plant cells in a number of different forms with different functions essential for plant life. Among them, chloroplasts are by far the more extensively studied owing to their central role in photosynthesis. However, other plastid family members, often referred to as nongreen plastids, play also major roles in the physiology of higher plants and could be better suited for studies of specific metabolic processes in heterotrophic plant cells. Unfortunately, serious technical problems are frequently encountered with separating intact, active nongreen plastids from contaminating membranes and mitochondria. Here, we provide detailed protocols suitable for the large scale preparation of intact and highly pure proplastids from cauliflower buds, as well as amyloplasts from sycamore cultured cells, and for the subsequent separation of their surrounding envelope membranes from the stroma and other plastid fractions. Both methods proved to be highly reliable and have been instrumental for in-depth investigations on biochemistry and physiology of nongreen plastids.

Mechanism of Nutrient Deprivation Induced Triacylglyceride Accumulation in Alga Indicated by Fluorescence Hyperspectral Imaging

Microalgae have been identified as a promising renewable feedstock for production of lipids for feeds and fuels. For a wide variety of algal species, depletion of major nutrients from the growth medium results in dramatically increased biosynthesis of triacylglycerides, in some cases exceeding 70% of the dry weight of the biomass. In recent work, we explored this process in a number of promising algal production strains (Chlorophyceae and Eustigmophyceae) using hyperspectral confocal fluorescence imaging with multivariate spectral unmixing. By coupling intensity modulation of the excitation laser and attenuation of the dominant chlorophyll fluorescence emission, we identified a distinct spectral component composed of chlorophyll fluorescence and resonance Raman emission for lipid-solubilized carotenoids allowing label-free projections of lipid bodies in addition to plastids. Furthermore, cells exposed to the lipophilic dye, Nile Red, exhibited distinct spectral components corresponding to the polar and neutral lipid fractions. Combining these capabilities for analysis of nutrient deprivation time courses revealed increases in the lipid fraction of the cellular volume of ∼500%, as well as a correlated decrease in the plastid fraction of the total cellular volume. Additionally, diffraction limited tubules connecting the lipids bodies to the plastids and the presence of unbound chlorophyll in the lipid bodies provided evidence for direct shuttling of lipids from the chloroplast to the lipid bodies, in opposition to the expected ER to lipid body transfer dictated by the Kennedy pathway. The results suggest that nutrient deprivation-based lipid accumulation in these species is dominated by plastid membrane recycling, independent of acyl-CoA. Finally, an additional spectral component exhibiting high spectral overlap with Nile Red and varying inversely with the chlorophyll emission was detected during senescence, potentially resulting in false positives for lipid enrichment in low spectral resolution assays.

The localization of nitrite reductase, glutamate synthase and malate metabolism enzymes in Pisum arvense L. roots

Centrifugation of a homogenate made from <em>Pisum arvense</em> L. roots in a sucrose density gradient enabled the separation of the plastid fraction from mitochondria and microsomes. The presence of nitrite reductase and glutamate synthase was demonstrated in the plastids. Malic enzyme activity was not linked with any organelle fraction and was found only in the cytosol. High malate dehydrogenase activity was found in the mitochondria fraction, although its activity was also determined in plastids. The results suggest that malic acid metabolism in plastids may be the source of reduced pyridine nucleotides for reactions catalysed by nitrite reductase and glutamate synthase.

Biochemical properties and enzymic capacities of chromoplasts isolated from wild buttercup ( Ranunculus acris L.)

Chromoplasts were extracted from the perianth of the wild Meadow Buttercup ( Ranunculus acris L.) and purified by sucrose density gradient centrifugation. Analysis of marker enzymes indicated that chromoplast preparations were essentially free of contamination by cytosol, mitochondria and microbodies. The distribution and recovery of glycolytic enzymes indicated an incomplete sequence of enzymes for this pathway in chromoplasts. Fructose 1, 6- bisphosphate aldolase (aldolase) was the only glycolytic enzyme for which unequivocal evidence of localisation within chromoplast preparations could not be obtained. Sub-cellular fractionation and immunological studies using antibodies raised against spinach cytosolic and chloroplastic aldolase, revealed that over 40% of its activity was associated with a washed particulate/membrane fraction in chromoplasts, but this could be removed with 1.2 M NaCl. It is concluded that Ranunculus chromoplasts do not possess a plastidial form of aldolase, and that the aldolase measured in the plastid fraction is a cytosolic form associating with plastid membranes. Isolated buttercup chromoplasts possess a functional oxidative pentose-phosphate pathway (OPPP) which could be monitored as 14CO 2 release following incubation with [U- 14C]-glucose 6-phosphate (Glc6P). 14CO 2 was not evolved when [U- 14C]-glucose 1-phosphate (Glc1P) was supplied to chromoplasts suggesting this compound is not transported into the intact organelle and that no interconversion of hexose-phosphate by phosphoglucomutase occurred outside the plastid. Transport experiments using native chromoplast vesicles preloaded with inorganic orthophosphate indicated that these organelles transported Glc6P and dihydroxyacetone phosphate (DHAP), but not Glc1P. The chromoplasts isolated from this wild plant have similar enzymic components and transport properties to other chromoplasts studied to date.

PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants.

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.

ATP citrate lyase in the glaucocystophyte alga Cyanophora paradoxa is a cytosolic enzyme: characterisation of the gene for the large subunit at the cDNA and genomic levels.

The single-copy gene for the large subunit of ATP citrate lyase (ACL) from the alga Cyanophora paradoxa was characterized at the cDNA and genome levels. The gene product showed high sequence similarity to its mammalian counterparts, but is smaller in size, as is also found for the fungal subunit Acl1 and, most probably, for the corresponding subunit from Arabidopsis thaliana. The C. paradoxa gene is interrupted by at least 12 introns of 53-65 bp with conserved border and branchpoint sequences, and the product lacks a stroma-targeting peptide. Enzyme activity was found in the cytosol of C. paradoxa but not in the plastid (cyanelle) fraction. This is in contrast to the subcellular distribution of ATP citrate lyases in higher plants, where both chloroplast and cytosolic enzymes have been reported.

Brassica rapa has three genes that encode proteins associated with different neutral lipids in plastids of specific tissues.

Plastid lipid-associated protein (PAP), a predominant structural protein associated with carotenoids and other non-green neutral lipids in plastids, was shown to be encoded by a single nuclear gene in several species. Here we report three PAP genes in the diploid Brassica rapa; the three PAPs are associated with different lipids in specific tissues. Pap1 and Pap2 are more similar to each other (84% amino acid sequence identity) than to Pap3 (46% and 44%, respectively) in the encoded mature proteins. Pap1 transcript was most abundant in the maturing anthers (tapetum) and in lesser amounts in leaves, fruit coats, seeds, and sepals; Pap2 transcript was abundant only in the petals; and Pap3 transcript had a wide distribution, but at minimal levels in numerous organs. Immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most organs had several nanograms of PAP1 or PAP2 per milligram of total protein, the highest amounts being in the anthers (10.9 microg x mg(-1) PAP1) and petals (6.6 microg x mg(-1) PAP2), and that they had much less PAP3 (<0.02 microg x mg(-1)). In these organs PAP was localized in isolated plastid fractions. Plants were subjected to abiotic stresses; drought and ozone reduced the levels of the three Pap transcripts, whereas mechanical wounding and altering the light intensity enhanced their levels. We conclude that the PAP gene family consists of several members whose proteins are associated with different lipids and whose expressions are controlled by distinct mechanisms. Earlier reports of the expression of one Pap gene in various organs in a species need to be re-examined.

Lipoxygenase pathway in olive callus cultures (Olea europaea)

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereo-specific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.

The chloroplast bchI gene encodes a subunit of magnesium chelatase in the marine heterokont alga Heterosigma carterae

The bchI gene (synonym: chlI), whose product is involved in magnesium chelatase activity, is located in the small single-copy region of the plastid genome of Heterosigma carterae (heterokont alga, Raphidophyceae; formerly named Olisthodiscus luteus). As a unique feature, bchI in H. carterae overlaps an upstream open reading frame (ORF97) of unknown function by 23 base-pairs. Cells contain mRNAs for the full (1.6 kb) cotranscript as well as transcripts for bchI (1.2 kb) and ORF97 (0.4 kb). Transcription initiation in a plastid run on assay of the ORF97/bchI gene cluster is approximately 5-fold higher in cultures sampled in the light versus the dark. In contrast, Northern analysis shows that all three transcripts are in equal abundance at both light and dark sampling times. An antibody raised against a BchI fragment expressed in Escherichia coli recognized a protein of the expected size in the plastid fraction of H. carterae on a Western blot. Analysis of BchI amino acid sequences suggests that the protein may be membrane translocated and could bind ATP. Phylogenetic analysis of bchI sequences documents a deep evolutionary branching between chlorophyll a/b and non-chlorophyll b plastids.

The chloroplast bchI gene encodes a subunit of magnesium chelatase in the marine heterokont alga Heterosigma carterae

The bchI gene (synonym: chlI), whose product is involved in magnesium chelatase activity, is located in the small single-copy region of the plastid genome of Heterosigma carterae (heterokont alga, Raphidophyceae; formerly named Olisthodiscus luteus). As a unique feature, bchI in H. carterae overlaps an upstream open reading frame (ORF97) of unknown function by 23 base-pairs. Cells contain mRNAs for the full (1.6 kb) cotranscript as well as transcripts for bchI (1.2 kb) and ORF97 (0.4 kb). Transcription initiation in a plastid run on assay of the ORF97/bchI gene cluster is approximately 5-fold higher in cultures sampled in the light versus the dark. In contrast, Northern analysis shows that all three transcripts are in equal abundance at both light and dark sampling times. An antibody raised against a BchI fragment expressed in Escherichia coli recognized a protein of the expected size in the plastid fraction of H. carterae on a Western blot. Analysis of BchI amino acid sequences suggests that the protein may be membrane translocated and could bind ATP. Phylogenetic analysis of bchI sequences documents a deep evolutionary branching between chlorophyll a/b and non-chlorophyll b plastids.

Subcellular location of polyphenol oxidase in apples.

The location of polyphenol oxidase (PPO) in cells of apple fruit was examined by immunohistochemistry and subcellular fractionation. In mature apple fruits, where vacuoles occupy most of the cells, PPO was detected immunochemically near the cell walls with use of anti-apple PPO antibodies. In cells of immature fruits and tissue culture, PPO was detected in organelles other than the vacuoles, probably in plastids. The plastid fraction was purified by density gradient ultracentrifugation, and the activities of PPO and marker enzymes of plastids were the highest in the plastids. Most apple PPO was in plastids, as are other plant PPOs, and some of the protein was solubilized and proteolyzed during ripening and storage.

Ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [14C]geranylgeranyl diphosphate to ent-[14C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [14C]geranylgeranyl diphosphate into ent-[14C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[14C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.

In-situ localization of nitrate reductase in maize roots

The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.

Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme, in Citrus fruit peel.

Chlorophyllase (Chlase; EC 3.1.1.14) was extracted from plastid fractions of ethylene-treated orange fruit peel and purified 400-fold to homogeneity by gel filtration, hydrophobic chromatography, and preparative SDS/PAGE of nonheated protein. SDS/PAGE of nonheated purified enzyme indicated that Chlase activity is associated with a single protein band migrating at an apparent molecular mass of 25 kDa whereas the heated purified enzyme had a molecular mass of 35 kDa. The N-terminal sequence of the purified protein was determined. The purified enzyme was used as an immunogen for raising antibodies in rabbits. The antiserum was highly specific and on Western blots recognized both the heated and the nonheated form of Chlase. The antibodies also recognized the solubilized enzyme, as shown by an immunoprecipitation assay and by antigen-antibody capture assays in microtiter plates. Treatment with ethylene, which enhances degreening, increased Chlase activity 12-fold. Immunoblot analyses of crude extracts from ethylene-treated fruit detected a strong signal of the Chlase protein, while only a trace level of the enzyme protein could be detected in air. Gibberellin A3 and N6-benzyladenine partly counteracted the ethylene-induced increase in Chlase activity as well as the immunodetected upsurge of the Chlase protein. Ethylene appears to enhance the degreening of citrus fruit through de novo synthesis of the Chlase protein, which in turn is inhibited by the senescence-delaying regulators, gibberellin A3 and N6-benzyladenine. The Chlase enzyme protein may, therefore, serve as a model system for studying the hormonal molecular regulation of fruit ripening and senescence.

Isolation and Characterization of a Chlamydomonas reinhardtii Mutant Resistant to Photobleaching Herbicides

A group of highly active N -phenylimide photobleaching herbicides have been synthesized. These N -phenylimide herbicides as well as diphenyl ether herbicides induce protoporphyrin IX accumulation and inhibit protoporphyrinogen oxidase activity at extremely low concentrations in higher plants. The binding of a 14C -labeled N -phenylimide herbicide S-23121 [N-[4-chloro- 2-fluoro-5-[(1-m ethyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubilized plastid fractions of greening corn seedlings is competed by the diphenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibitor of photosynthetic electron transport. These results indicate a similar mode of action for both N -phenylimide and diphenyl ether herbicides. In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 resistant to N -phenylimide S-23142 [N -(4-chloro- 2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] was isolated by mutagenesis with N -m ethyl-N′-nitro-N -nitrosoguanidine. The 90% inhibition concentration of N -phenylimide S-23142 for growth of RS-3 was 100 times higher than that for wild type. Maximum accumulation of protoporphyrin IX was reached at 0.03 μᴍ of S-23142 for the wild type and 3 μᴍ for RS-3. RS-3 was resistant to oxadiazon, oxyfluorfen and acifluorfen-ethyl which had been shown to have the same mechanism of action as N -phenylimide herbicides, but not to paraquat, diuron or fluridone. Genetic analysis of RS-3 strain showed that the resistance results from a dominant mutation ( rs-3) in the nuclear genome. The magnesium protoporphyrin IX synthesizing activity from 5-am inolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporphyrinogen oxidase activity in Percoll™ -purified chloroplasts from RS-3 was also less sensitive to S-23142 than that from wild type. These results indicate that the resistance of RS-3 is specific for photobleaching herbicides, and that the mutation is related to protoporphyrinogen oxidase, the primary site of the photobleaching herbicide action.

Lipid changes in microsomes and crude plastid fractions during storage of tomato frutis at chilling and nonchilling temperatures

Mature-green tomato fruits ( Lycopersicon esculentum) were stored for four or 12 days at chilling (2°) or nonchilling (15°) temperature. Fruits stored for 12 days at 15° ripened to the turning stage, whereas fruits at 2° did not ripen. Lipids of plastid and microsomal membrane fractions from pericarp tissue were analysed at harvest and after four or 12 days of storage. After 12 days at either 15° or 2°, the ratio of phospholipids (PL) to protein in microsomes declined, with a concomitant increase in the ratios of total membrane sterols (TMS) and cerebrosides (CB) to PL. The TMS:PL and CB:PL ratios also increased in crude plastid fractions. In both microsomes and plastids, free sterols (FS) increased more at 2° than at 15°, and hence accounted for a larger percentage of the TMS (FS + acylated steryl glycosides + steryl glycosides). The ratio of stigmasterol to sitosterol in all steryl lipids, but particularly in FS, increased more at 15° than at 2°. The unsaturation index of fatty acids in PL and galactolipids generally increased slightly during storage at both 15° and 2°. The ratio of phosphatidylethanolamine to phosphatidylcholine increased in both membrane fractions at both temperatures. In plastids, the ratio of mono- to digalactosyldiacylglycerols declined substantially at 2° but not at 15°.

Subcellular Location of Δ1-Pyrroline-5-Carboxylate Reductase in Root/Nodule and Leaf of Soybean

The expression of Delta(1)-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated (AJ Delauney, DPS Verma [1990] Mol Gen Genet 221: 299-305). P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCl). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate(+) inhibited P5CR activity, whereas nicotinamide adenine dinucleotide(+) did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production.

Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila).

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.

In vitro mode of action of N-Phenylimide photobleaching herbicides

The effect of N-Phenylimide photobleaching herbicides on in vitro synthesis of protoporphyrin was examined. The N-phenylimide photobleaching herbicide S-23142 [ N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] inhibited the Mg-protoporphyrin IX synthesis of intact plastids by 50% at 10 −10 M. Protoporphyrin IX synthesizing activity was solubilized from plastid membranes by n-dodecyl β- d-maltoside, and the solubilized activity was inhibited by several photobleaching herbicides. In vitro binding of N-phenylimide photobleaching herbicide, S-23121 [ N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxyl]phenyl]-3,4,5,6-tetrahydrophthalimide], to a solubilized plastid fraction was examined. A good correlation between protoporphyrin IX synthesizing activity and [ 14C]S-23121 specific binding was observed. Binding site affinity ( K d ) was 8.9 – 9.8 n M. The binding was displaced by a diphenylether photobleaching herbicide, acifluorfenethyl, and another N-phenylimide, S-23142, but not by a photosystem II electron transport inhibitor, DCMU. Bound [ 14C]S-23121 was dissociated by an excess of cold S-23121, which indicated the reversible binding of S-23121.