Polyphenol oxidase: The chloroplast oxidase with no established function

Vaughn, K. C, Lax, A. R. and Duke, S. O. 1988. Polyphenol oxidase: The chloroplast oxidase with no established function. - Physiol. Plant. 72: 659–665.
Polyphenol oxidase (PPO) is an enzyme localized on the thylakoids of chloroplasts and in vesicles or other bodies in non-green plastid types. Although virtually all plastids contain PPO, little or no detectable activity is associated with guard cell and bundle sheath cell chloroplasts. Despite this nearly ubiquitous occurrence, no function for this enzyme has been established. The enzyme is nuclear-encoded and, unlike most chloroplast proteins is not encoded as a larger M, precursor molecule. This lack of a transit peptide sequence may be related to a unique mechanism of uptake, apparently involving inner envelope-derived vesicles. The M, range of most of the PPO forms is 36–45 kDa. PPO is apparently not involved in phenolic biosynthesis but is probably involved with the production of o -quinones during pathogen invasion. A role for PPO as an "oxygen buffer" is postulated, but little concrete data have been collected on any other functional role for this enzyme.     

Chloroplast proteins of cytoplasmic origin. Effect of chloramphenicol on their synthesis, transport and intraplastidial localization. Ultrastructural autoradiographic studies

Ultrastructural autoradiographic studies after application of 3H-lysine indicate that during the transformation of the etioplasts into chlorplasts in the bean (Phaseolus vulgaris) the protein synthesis in plastids occurs mainly near the thylakoid membranes and the prolamellar bodies: most of the autoradiographic grains are placed over the structures. After 24 h postincubation in nonradioactive medium the ratio of the number of silver grains associated with thylakoids to those over stroma increases more than 2.5 times in control plants, whereas in cells treated with chloramphenicol only 1.5 times. Simultaneously in chloramphenicol-treated plants an increase in plastid envelope labelling is observed. It has been assumed that chloramphenicol, having no inhibitory effect on the synthesis and transport of proteins imported from the cytoplasm to the plastid, lowers their penetration inside the plastid as well as their incorporation into the thylakoid membrane.     

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.     

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.     

Localization of the enzyme geranylgeranylpyrophosphate synthase in Capsicum fruits by immunogold cytochemistry after conventional chemical fixation or quick-freezing followed by freeze-substitution. Labelling evolution during fruit ripening

The enzyme geranylgeranylpyrophosphate synthase (GGPPS), which plays a key role in the synthesis of diterpene compounds, carotenoids and higher terpenoids, has been localized in Capsicum fruit cells by ultrastructural immunogold cytochemistry, after conventional chemical fixation of tissues and quick-freezing followed by freeze-substitution of isolated chloroplasts and chromoplasts. In agreement with previous biochemical studies on cell fractions, the enzyme seems restricted to the plastid compartment. Together with the phenotypic changes of the fruit and the ultrastructural modifications of the plastids during the transition of chloroplasts to chromoplasts, the amount of immunolabelling over plastid sections increases more than a ten-fold factor in the course of fruit ripening. In chemically fixed tissues, the gold labelling of chloroplasts is very faint and erratically localized whereas in further transition stages, and in chromoplasts, most of the gold particles surround the developing plastoglobuli, which are the characteristic carotenoid-bearing structures. Because of the very low and inconstant labelling of chloroplasts in green fruits after chemical fixation, cryofixed and acetone freeze-substituted purified plastids were used as a model system for an accurate localization of the enzyme in these organelles. Quick-freezing in buffered sucrose by slam-freezing on a cold copper block results in optimal preservation of the plastids and improved labelling of GGPPS. The enzyme is not scattered at random throughout the stroma. Gold particles are concentrated in distinct stroma regions, and especially at the sites of initiation of stroma globuli which are the early structural event of carotenoid accumulation. A few gold particles are also present on the margins of thylakoids and, presumably, on the plastid envelope. This paper reports further evidence of the central role of the plastid compartment in the production of C20 isoprenoid intermediates in the plant cell, shows the spatial relationship of the enzyme geranylgeranylpyrophosphate synthase with the plastid substructures and the existence of several GGPPS pools within the plastids. It demonstrates the interest of cryo-methods for an accurate localization of various enzymes in plant cells.     

Tissue localization of polyphenol oxidase inSorghum

Summary Plastidic polyphenol oxidase (PPO) was localized in various plastid types ofSorghum bicolor (L.) Moench using cytochemical and biochemical franctionation techniques. PPO was found to be present in the mesophyll plastids yet absent from the bundle sheath and guard cell plastids. Mechanical fractionation of mesophyll and bundle sheath plastids, with subsequent electrophoretic or spectrophotometric assay of the preparations, also indicated that PPO was absent from the bundle sheath but present in the mesophyll fraction. A developmental study revealed that, although all leaf plastids near the basal meristem were ultrastructurally similar, the mesophyll and bundle sheath plastids were already differentiated with respect to PPO activity.     

Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants

Two chloroplastic proteins, named CDSP 32 and CDSP 34 for chloroplastic drought-induced stress protein of 32 and 34 kDa, were previously shown to be substantially synthesized in Solanum tuberosum plants subjected to water deficit. We investigated the localization of CDSPs in leaf chloroplasts from control and wilted potato plants using immunocytochemistry. Observation of electron micrographs did not reveal any important change in plastid structures of drought-stressed plants, except an increased number and a larger size of plastoglobuli. In well-watered plants, very little labeling corresponding to CDSP 32 was detected. Consecutively to water stress, a higher abundance of CDSP 32 was revealed, the protein being exclusively localized in the stroma. Immunocytochemical data indicated the presence of some CDSP 34 protein in well-watered plants and confirmed its accumulation upon water deficit. CDSP 34 was found to be preferentially associated with stromal lamellae thylakoids, but some protein was revealed in the stroma. No association of CDSP 34 with grana and plastoglobuli was noticed in chloroplasts from control and stressed plants.     

Isolation of a full-length cDNA encoding polyphenol oxidase from sugarcane, a C4 grass

Polyphenol oxidase (PPO) activity in sugarcane (a C4 grass) was highest in the growing point and declined down the stalk. Sugarcane PPO with an apparent molecular mass of 45 kDa was purified to homogeneity from immature stem tissue. Western analysis of sugarcane extracts with a polyclonal antibody raised to this protein suggested it resulted from cleavage of a 60 kDa protein during purification. The antibody was used to screen a sugarcane stem cDNA library. A full-length PPO clone (sugppol) was characterised and shown to encode a 67 kDa precursor protein comprising a plastid transit sequence of 8 kDa and a mature PPO protein of 59 kDa. High levels of expression ofsugppol were detected in the growing point of the stalk and in the immature tissue immediately below it, but no message was detected in RNA from mature stem or leaf. Comparison with other PPO sequences indicated thatsugppol was significantly different to PPO genes in C3 dicotyledonous plants.     

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.     

Import, targeting, and processing of a plant polyphenol oxidase.

A tomato (Lycopersicon esculentum L.) gene encoding a precursor of polyphenol oxidase (PPO) was transcribed and translated in vitro. The import, targeting, and processing of the [35S]methionine-labeled precursor protein (pPPO) were studied in isolated chloroplasts. The protein was routed to the thylakoid lumen in two steps. The 67-kD precursor was first imported into the stroma in an ATP-dependent step. It was processed to a 62-kD intermediate by a stromal peptidase. Translocation into the lumen was light dependent and involved processing of the 62-kD to the 59-kD mature form. The mature polypeptide was soluble in the lumen and not bound to thylakoids. This two-step targeting pattern was observed in plastids from a variety of plants including pea (Pisum sativum L.), tomato, and maize (Zea mays L.). The ratio between the intermediate and mature forms observed depended on the plant species, leaf age, growth conditions, and illumination regime to which the plants had been subjected. Cu2+ was not required for pPPO import or processing. Furthermore, low concentrations of Cu2+ (1-5 microM) markedly inhibited the first import step. Tentoxin specifically inhibited pPPO import, leaving the precursor bound to the envelope membrane. The two-step routing of pPPO into chloroplasts, typical of thylakoid lumen proteins, is consistent with the two-domain structure of the transit peptide and appears to be a feature of all plant PPO genes isolated so far. No evidence was found for unorthodox routing mechanisms, which have been suggested to be involved in the import of plant PPOs. The two-step routing may account for some of the multiplicity of PPO observed in vivo.     

The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron

The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previously described to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1 knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid development and plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function in protection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered from oxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants were characterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 protein increase in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of other transition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency, suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading. In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated in PIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. In consequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particular fruit development.     

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.     

A Dithiothreitol-Sensitive Tetrameric Protease from Spinach Thylakoids Has Polyphenol Oxidase Activity

Polyclonal antibody raised against a dithiothreitol-sen-sitivetetrameric protease (DSTP) from PSII membranes specificallyinhibited the polyphenol oxidase (PPO) activity of spinach thylakoids.DSTP was copurified with PPO activity on an affinity columnprepared with antibody against DSTP. These results suggest thatDSTP and PPO are the same protein. During purification of DSTP,Tween 20 was essential for stabilization of the protein, whichwas degraded in the absence of the detergent. Gel-filtrationchromatog-raphy of the purified DSTP revealed the presence of230-kDa (tetramer) and 60-kDa (monomer) species. The coppercontent of monomer species was determined to be 0.4 Cu atomper protein molecule, when the molecular weight of the proteinwas calculated to be 62,243, which is the value reported forspinach PPO [Hind et al. (1995) Biochemistry 34: 8157]. PurifiedDSTP caused the degradation as well as the dimerization of theextrinsic 23-kDa protein of PSII. The degradation of the proteinwas suppressed under anaerobic conditions induced by the presenceof glucose oxidase and glucose together. This fact suggeststhat oxygen molecules are involved in the proteolytic reactionand that the proteolytic activity and PPO activity may be correlatedwith each other. (Received September 27, 1996; Accepted December 4, 1996)     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

Visualization of plastid nucleoids in situ using the PEND-GFP fusion protein

Plastid DNA is a circular molecule of 120-150 kbp, which is organized into a protein-DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND-green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND-GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.     

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.     

Non-vesicular and vesicular lipid trafficking involving plastids

In plants, newly synthesized fatty acids are either directly incorporated into glycerolipids in the plastid or exported and assembled into lipids at the endoplasmic reticulum (ER). ER-derived glycerolipids serve as building blocks for extraplastidic membranes. Alternatively, they can return to the plastid where their diacylglycerol backbone is incorporated into the glycerolipids of the photosynthetic membranes, the thylakoids. Thylakoid lipids are assembled at the plastid envelope membranes and are transferred to the thylakoids. Under phosphate-limited growth conditions, galactolipids are exported from the outer plastid envelope membranes to extraplastidic membranes. Proteins, such as TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1) or VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), which are involved in different aspects of plastid lipid trafficking phenomena have recently been identified and mechanistic models that are based on the analysis of these components have begun to emerge.     

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.     

Plastid Alternative Oxidase (PTOX) Promotes Oxidative Stress When Overexpressed in Tobacco

Photoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b₆f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant system.