Function of polyphenol oxidase in higher plants

Recent evidence has supported the folllowing views:
1. Polyphenol oxidase (PPO) is a plastidic enzyme that is unclear-coded, but is inactive until incorporated into the plastid.
2. In healthy green tissues PPO exists in a latent form on the thylakoid membrane and is not involved in synthesis of phenolic compounds. In leucoplasts, proplastids, or amyloplasts PPO is often present in a latent form in rudimentary thylakoids.
3. PPO normally functions as a phenol oxidase in vivo only in sencent or damaged cells.
4. In the functional chloroplast, PPO may be involved in some aspect of oxygen chemistry – pherhaps mediation of pseudocyclic photophosphorylation.     

Tentoxin-induced loss of plastidic polyphenol oxidase

Tentoxin-treated mung bean plants are shown to lack chloroplast polyphenol oxidase (PPO) by enzymatic, electrophoretic and cytochemical analysis. Incorporation of PPO (a protein coded by nuclear DNA) into the plastid may occur via concentration of the protein into inner envelope-derived vesicles. PPO integration into the plastid is apparently blocked by a tentoxin treatment although fraction I protein (and hence the proteins for chloroplast ribosome production) is not affected by this fungal toxin. Both apical and etiolated plastids from teotoxin-treated plants lack PPO. Thus, it is unlikely that the primary effect of tentoxin is due to the binding of the chloroplast coupling factor, as previously supposed.     

Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in Chlamydomonas reinhardtii

Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.     

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.     

Polyphenol oxidase and photosynthesis research

Very briefly, the present state of knowledge on the latent, lumen oriented polyphenol oxidase (PPO) of the chloroplast is reviewed. The location of PPO in the thylakoid membrane was described by D. Arnon 46 years ago. The N-terminus sequence of the spinach enzyme is reported. A historical sketch is given of the discovery of photophosphorylation and Arnon's visit to the admired O. Warburg.     

Import of polyphenol oxidase by chloroplasts is enhanced by methyl jasmonate

Polyphenol oxidase (PPO; EC 1.10.3.2 or EC 1.14.18.1) takes part in the response of tomato plants (Lycopersicon esculentum Mill.) to wounding and herbivore attack, mediated by the octadecanoid wound-signaling pathway. Wounding and methyl jasmonate (MeJA) induce expression of ppo genes and markedly increase the level of the enzyme. We report that pretreatment with MeJA also markedly increased the ability of isolated tomato chloroplasts to import and process PPO precursors (pPPO). Pea (Pisum sativum L.) chloroplasts showed no such response. Wounding or ethylene alone was ineffective but ethylene was synergistic with MeJA. Treatment with MeJA conferred a strong binding of pPPO, or its processing intermediate, to thylakoids and subsequent translocation into the lumen and processing to the mature protein. The effect on PPO import and translocation was evident after 8–16 h exposure to MeJA. Membrane-bound pPPO was cross-linked to a proteinaceous component of the thylakoid translocation apparatus, apparently induced by MeJA. The import and processing of other nuclear-encoded thylakoid proteins were not affected by MeJA in tomato. A 90-kDa protein that co-fractionated with thylakoids was induced along with the increase in competence for PPO import, and was identified as the proteinase-inhibitor multicystatin. It is concluded that the 90-kDa protein observed is part of the MeJA-induced defense response of tomato, not a component of the thylakoid translocation apparatus.Abbreviations Chl Chlorophyll - i and p Prefixes used to denote the intermediate and precursor forms of a protein, respectively - JA Jasmonic acid - LSU Large subunit of Rubisco - MeJA Methyl jasmonate - OE23 and OE33 23- and 33-kDa subunits of the oxygen-evolving complex of PSII - PC Plastocyanin - pPPO (iPPO, PPO) Precursor (intermediate, mature) form of polyphenol oxidase     

Tentoxin stops the processing of polyphenol oxidase into an active protein

Previous studies in our laboratory have indicated that polyphenol oxidase (PPO), as measured by its activity, is not present in tentoxin-treated plants. In the present study, immunochemical techniques were used as a sensitive probe of tentoxin effects on PPO. Immunocytochemistry of PPO with peroxidase-antiperoxidase labelling techniques, revealed that in control Vicia faba L. chloroplasts, PPO was associated mainly with the thylakoids. Cytochemical staining of PPO activity using DL-dihydroxyphenylalanine (DOPA) as substrate was also localized only on the thylakoids in the control chloroplasts. In tentoxin-treated plants all of the immunologically-recognizable PPO accumulated at the plastid envelope although no PPO was detected by cytochemical methods. SDS polyacrylamide gels of extracts from control and tentoxin-treated Vicia leaves were blotted onto nitrocellulose and reacted with rabbit anti-PPO. Secondary labelling of the blots with goat-antirabbit IgG labelled with peroxidase revealed a 40 kdalton protein in both extracts. However, only the PPO from the control extract had DOPA oxidase activity. These data suggest that PPO accumulates in the plastid envelope membranes in tentoxin-affected cells and that PPO in these treated plants is not processed to an active protein.     

The fine structure of Synura sphagnicola (Korsh.) Korsh. (Chrysophyceae)

S. sphagnicola resembles other species of Synura previously described by electron microscopy in most features of structure but differs in possessing pyrenoids and up to five cylindrical stacks of smooth cisternae which occur between the pyrenoids and leucosin vesicles. Each stack is surrounded by a tubular cisterna which bears ribosomes on its distal face but there are no clear permanent connections between this and the chloroplast ER. Other features apparently unique to this species previously known from light microscopy are described. These include the axial position of the chloroplasts; the peripheral position of the leucosin vesicles; and the loose attachment of the scales. The structure of the body scales is described for the first time from sections. The flagellar scales are formed in the swollen edges of the Golgi cisternae and appear to pass to the cell surface in large vesicles.     

鸭梨PPO与GFP融合基因的烟草转化及亚细胞定位观察

将鸭梨PPO基因与绿色萤光蛋白GFP基因相融合共同进行遗传转化的方式,对鸭梨多酚氧化酶开展细胞定位研究。通过克隆该酶基因除终止密码子TAA外长度为1 779bp的CDS序列,与绿色荧光蛋白基因重组构建了荧光表达载体pBI121-PPO-GFP,借助农杆菌转化烟草,转基因烟草叶片细胞经激光扫描共聚焦显微镜观察,绿色荧光蛋白荧光与叶绿体自发荧光相重合。结果表明鸭梨多酚氧化酶为叶绿体蛋白质。     

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.     

Response of mitochondrial alternative oxidase (AOX) to light signals

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes energy wasteful cyanide (CN)-resistant respiration and plays a role in optimizing photosynthesis. Recent studies from our group indicated that AOX plays a crucial role in chloroplast protection under extreme environments, such as high light (HL). Genetic data suggest that AOX is upregulated by light that was mediated by photoreceptors (phytochromes, phototropins and cryptochromes), and it also might have a particular role in relieving the overreduction of chloroplasts. Physiological analyses further suggest that AOX is essential for the dark-tolight transition, especially in the course of de-etiolation. In this mini-review, we highlight recent progress in understanding the beneficial interaction between photosynthesis and mitochondria metabolism and discuss the possible role and mechanism of AOX in dissipation of excess reduced equivalents for chloroplasts under high light condition.Key words: alternative oxidase (AOX), excess light, NAD(P)H dehydrogenases (NDs), photoreceptors, reactive oxygen species (ROS)     

Binding of pyruvate oxidase alpha-peptide to phospholipid vesicles

The alpha-peptide of pyruvate oxidase is a 23 residue peptide which is cleaved from the carboxy terminus of the enzyme during proteolytic activation by chymotrypsin (M. Recny et al. (1985) J. Biol. Chem. 260, 14287-14291). Cleavage of alpha-peptide results in the loss of the high affinity lipid-binding site in the enzyme. The beta-peptide of pyruvate oxidase is a 101 residue peptide which also is cleaved from the carboxy terminus of pyruvate oxidase. Cleavage of the beta-peptide from pyruvate oxidase results in the inactivation of the enzyme. The beta-peptide includes the alpha-peptide amino acid sequences at its carboxyl terminus. We now report on the binding of the alpha- and beta-peptides to phospholipid vesicles. Both peptides bind with equal and high affinity to phosphatidylcholine vesicles. We conclude from these results that the alpha-peptide furnishes the membrane-binding site which plays the physiologically important role in the activation of this peripheral membrane enzyme.     

STUDIES ON OXALIC ACID OXIDASE IN GREEN LEAVES

1. The leaves of Chenopodium ambrosioides L. were found to havean intense activity of oxalic acid oxidase. The enzyme was locatedin the chloroplast, being firmly hound to its structure. 2. Properties of this chloroplast oxalic acid oxidase were described.The strict aerobic nature and the stoichiometry of the reactionwere confirmed. 3. Isolation of the enzyme from chloroplasts was performed,rupturing the chloroplasts with a French pressure cell or usingpyridine-water (1:1) as an extracting medium. 4. The enzyme was found to contain flavine and its activitywas enhanced in the presence of flavine added. Accordingly,the enzyme was inferred to be a flavine enzyme. (Received December 23, 1963; )     

Some kinetic properties of polyphenol oxidase obtained from dill (Anethum graveolens)

Polyphenol oxidase (PPO) was partially purified from dill by (NH4)(2)SO4 precipitation followed by dialysis and gel filtration chromatography. Polyphenol oxidase activity was measured spectrophotometrically at 420 nm using catechol, dopamine and chlorogenic acid as substrates. Optimum pH, temperature, and ionic strength were determined with three substrates. The best substrate of dill PPO was found to be chlorogenic acid. Some kinetic properties of the enzyme such as V(max,) K(M) and V(max)/K(M) were determined for all three substrates. The effects of various inhibitors on the reaction catalysed by the enzyme were tested and I(50) values calculated. The most effective inhibitor was L-cysteine. Activation energies, E(a), were determined from the Arrhenius equation. In addition, activation enthalpy, DeltaH(a), and Q(10) values of the enzyme were also calculated.     

Polyphenol oxidase activity in the roots of seedlings of Bromus (Poaceae) and other grass genera

? Premise of the study: Phenolic compounds exuded by roots have been implicated in allelopathic interactions among plants. Root enzymes that destroy phenolics may protect plants against allelopathic inhibition and thus may aid in invasiveness. Phenolic-degrading enzymes are chiefly found in aboveground plant parts, but have also been previously reported in root tissues where the enzyme's function is unknown. We explored phenolic oxidase activity in emerging roots of grasses in a survey across different grass genera; in particular, we aimed to test whether grasses of the genus Bromus, known for their large invasion potential, differ in this respect from other grass taxa. ? Methods: We assayed a range of grass genera commonly found in the United States for root enzyme activity with spectrophotometric assays of phenol oxidase activity using l-DOPA as the main substrate. ? Key results: In the survey of a grass genera, we discovered that roots of the genus Bromus contain large amounts of polyphenol oxidase (PPO) activity, while all other tested grass genera, even ones closely related to Bromus, did not. PPO was found to be present at germination and remained active throughout the life of the plant. Compared to other PPOs, the enzyme present in Bromus appears to have a narrow substrate range. ? Conclusions: The specific functions of the root PPO and the ecological ramifications of the special status of Bromus are not yet clear. The possibility that the enzyme plays a role in plant species interaction for bromes, a genus of grasses known to have high invasive potential, is raised.     

Differences in the activity of superoxide dismutase, polyphenol oxidase and Cu-Zn content in the fruits of Gordal and Manzanilla olive varieties

Activity of the enzymes superoxide dismutase (SOD) and polyphenol oxidase (PPO) as well as Cu-Zn content have been monitored during the thirteen weeks growth of both Gordal and Manzanilla olive variety fruits. These metalloenzymes, with Cu and Zn in the prostetic group, are involved in controlling the redox balance in the chloroplast environment. The results indicated that, under similar phenological and environmental conditions, there are periodic peaks of SOD activity in both varieties, followed by fluctuations in the copper content of the fruit. This was interpreted as a common and simultaneous response to situations of oxidative stress, and this response was more intense in the variety Gordal. The enzyme PPO showed an activity peak at start of growth and then practically disappeared. Thus, its activity cannot be correlated with situations of stress or with changes of Cu and Zn in the fruit.     

Polyphenol oxidases in plants and fungi: going places? A review

The more recent reports on polyphenol oxidase in plants and fungi are reviewed. The main aspects considered are the structure, distribution, location and properties of polyphenol oxidase (PPO) as well as newly discovered inhibitors of the enzyme. Particular stress is given to the possible function of the enzyme. The cloning and characterization of a large number of PPOs is surveyed. Although the active site of the enzyme is conserved, the amino acid sequence shows very considerable variability among species. Most plants and fungi PPO have multiple forms of PPO. Expression of the genes coding for the enzyme is tissue specific and also developmentally controlled. Many inhibitors of PPO have been described, which belong to very diverse chemical structures; however, their usefulness for controlling PPO activity remains in doubt. The function of PPO still remains enigmatic. In plants the positive correlation between levels of PPO and the resistance to pathogens and herbivores is frequently observed, but convincing proof of a causal relationship, in most cases, still has not been published. Evidence for the induction of PPO in plants, particularly under conditions of stress and pathogen attack is considered, including the role of jasmonate in the induction process. A clear role of PPO in a least two biosynthetic processes has been clearly demonstrated. In both cases a very high degree of substrate specificity has been found. In fungi, the function of PPO is probably different from that in plants, but there is some evidence indicating that here too PPO has a role in defense against pathogens. PPO also may be a pathogenic factor during the attack of fungi on other organisms. Although many details about structure and probably function of PPO have been revealed in the period reviewed, some of the basic questions raised over the years remain to be answered.     

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.     

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.