Characterisation and changes in the antioxidant system of chloroplasts and chromoplasts isolated from green and mature pepper fruits

Purification and characterisation of pepper ( Capsicum annuum L) chloroplasts and chromoplasts isolated from commercial green, red and yellow mature fruits were undertaken. Induction of the synthesis of several antioxidants in organelles isolated from mature fruits was found. The ultrastructure of organelles and the presence and activity of SOD isozymes and enzymes involved in the ASC-GSH cycle, together with the non-enzymatic antioxidant content and some oxidative parameters, were analysed. It was found that lipids, rather than proteins, seem to be a target for oxidation in the chromoplasts. The ascorbate and glutathione contents were elicited during differentiation of chloroplasts into chromoplasts in both red and yellow fruits. The activity of SOD and of components of the ASC-GSH cycle was up-regulated, suggesting that these enzymes may play a role in the protection of plastids and could act as modulators of signal molecules such as O₂^˙− and H₂O₂ during fruit maturation. The presence of an Mn-SOD in chromoplasts isolated from yellow pepper fruits was also investigated in terms of structural and antioxidant differences between the two cultivars.     

Chromoplast-Specific Proteins in Capsicum annuum

Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast-specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M_r of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species.     

Sweet pepper plastids: enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane

Chloroplasts or chromoplasts were purified from sweet-pepper (Capsicum annuum L. cv. Yolo Wonder) fruits and analysed with respect to their enzymic equipment, the transport properties across the envelope membrane, and for the presence of a functional oxidative pentose-phosphate pathway (OPPP). It was demonstrated that both types of plastid contain enzyme activities that allow glycolysis and OPPP. During the developmental conversion from chloroplasts to chromoplasts the activities of enzymes catalysing potentially rate-limiting reactions in glycolysis increased considerably. Most enzyme activities involved in the plastidic OPPP stayed constant or decreased during ripening, but transaldolase activity increased by more than 500%. To analyse whether pepper fruit chromoplasts are able to use exogenously supplied carbohydrates for the OPPP we measured the rate of ¹⁴CO₂ release after application of radioactively labelled precursors. Isolated pepper fruit chromoplasts used exogenously supplied [U¹⁴C]glucose- 6-phosphate (Glc6P) as a precursor for the OPPP. The metabolic flux through this pathway was stimulated by the presence of additional compounds which require reducing equivalents for further conversion, e.g. nitrite, or 2-oxoglutarate plus glutamine. The [¹⁴C]Glc6P-driven OPPP in isolated chromoplasts exhibited saturation with rising concentrations of Glc6P, reaching highest rates at an external concentration of about 2 mM. Exogenously given [U¹⁴C]glucose 1-phosphate (Glc1P)′ did not lead to a release of ¹⁴CO₂, indicating that this hexose phosphate is not taken up into the intact plastid. Using a proteoliposome system in which the envelope membrane proteins from sweet-pepper chromoplasts were functionally reconstituted we demonstrated that Glc6P is transported in counter-exchange with inorganic phosphate (P_i) or other phosphorylated intermediates. The Glc6P was taken up into proteoliposomes with an apparent K _m of 0.34 mM. Surprisingly, in contrast to tomato fruit plastids, isolated chromoplasts from sweet-pepper fruits do not possess a phosphate translocator allowing the uptake of Glc1P. Rising exogenous concentrations of dihydroxyacetone phosphate strongly inhibited the metabolic flux through the OPPP. This observation is discussed with respect to the presence of two phosphate translocator proteins in the envelope of sweet-pepper chromoplasts and with respect to possible metabolic changes occurring in heterotrophic tissues during development. Received: 24 April 1997 / Accepted: 16 June 1997     

Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Some Structural Changes During Ripening of Mangoes (Mangifera indica var. Alphonso) by Abscisic Acid Treatment

Abscisic acid at 10^–6 M concentration enhances ripeningof mangoes. The cells in the pulp of the fruit are large andparenchymatous and lose their integrity due to cell wall hydrolysisat the ripe stage. They contain abundant polysaccharides, consistingmainly of starch, which is degraded during ripening. The chloroplastsare transformed to chromoplasts containing red or yellow carotenoidpigment. Abscisic acid treatment enhances all of these processes.Mitochondria, on the other hand, retain their structural integritythroughout the ripening process in untreated and abscisic acid-treatedmangoes. Mangoes, ripening, abscisic acid, structure     

Fine Structure and Carotenoid Composition of the Fibrillar Chromoplasts of Asparagus officinalis L.

The spindle-shaped chromoplasts of the ripe fruit of Asparagusofficinalis developed from chloroplasts that lost grana andstroma lamellae and accumulated large-sized plastoglobules,most of which transformed into fibrils of exceptionally largediameter. The carotenoid concentration was high in ripe fruits(2520 µg g^–1 fresh wt) and consisted mainly of capsanthin,ß-carotene and zeazanthin. A. officinalis is onlythe third species reported to contain both capsanthin and capsorubin. The large diameter of both fibrils and their associated plastoglobulesenabled verification that flbrils pass through plastoglobules.Unlike most fibrillar chromoplasts, the plastoglobules remainedassociated with flbrils even in fully-ripe fruit, and a straightline relationship can be established between plastoglobule andfibril diameter. The presence of more than one fibril developingfrom a plastoglobule, and the occasional existence of fibrilstwisted helically around one another, is interpreted to meanthat fibril growth by lateral addition of microfibrils is unlikely. The microfibrillar ultrastructure of Asparagus flbrils was differentto that found for Capsicum fibrils, and these differences areillustrated in two models. These differences, however, do notnecessarily mean that the fibrils of each species are constructedof different protein sub-units, although the bonding betweensub-units is different.     

The development and ultrastructure of lycopene bodies in chromoplasts ofLycopersicum esculentum

Summary Lycopene bodies are developed in tomato chromoplasts at temperatures permitting synthesis of lycopene. Their appearance seems to be in correlation with the formation of special rigid membranes. These membranes were not observed in chromoplasts of tomatoes ripened at 32 C, a temperature under which no lycopene is synthesized. Electron diffraction patterns of isolated lycopene bodies showed that the bulk of such a body is a lycopene crystal.Similarities between lycopene bodies of the tomato fruits and carotene bodies of carrot roots lead to the conclusion that classification of chromoplasts into distinct categories is valid only for certain stages of the chromoplast life cycle.     

Microelectrophoretic and 9-Aminoacridine Fluorescence Study of the Surface Electrical Properties of Suspension Cultured Catharanthus roseus Cells and Isolated Protoplasts: Effects of Abscisic Acid Treatment

The effects of abscisic acid (ABA) treatments on the surfaceelectrical properties of cells and isolated protoplasts fromCatharanthus roseus cell suspension cultures were studied byelectrophoretic mobility and 9-aminoacridine (9AA) fluorescencemeasurements. The surface charge densities of the cells andprotoplasts estimated from electrokinetic data were –0.064Cm^–2and –0.048 C m^–2 respectively. These values wereclose to that estimated by 9AA fluorescence technique i.e.,–0.053 Cm^–2 for the cells and –0.041 Cm^–2for the isolated protoplasts accordingly. The net negative surfacecharge density decreased after application of 10 µM and50 µM ABA in both cells and protoplats, the more pronouncedeffect being observed at 10 µM ABA. When 100 µMABA was supplemented to the cell suspension culture the oppositeeffect was observed. The average charge density increased to–0.074 C m^–2 for the cells, and to –0.055C m^–2 for protoplasts, as revealed from the 9AA measurements.The results are discussed in terms of specific concentrationdependent ABA-induced alterations of the electrostatic propertiesof cell and protoplast membranes. (Received December 12, 1994; Accepted April 3, 1995)     

Long-term regulation of contractility and calcium current in smooth muscle

Longitudinal smooth muscle strips from guinea pig ileum werecultured in vitro for 5 days, and the relationship betweenextracellular Ca²⁺ and force inhigh-K⁺ medium was evaluated. Instrips cultured with 10% fetal calf serum (FCS), this relationship wasshifted to the right (50% effective concentration changed by 2-3mM) compared with strips cultured without FCS. The shiftwas prevented by inclusion of verapamil (1 µM) during culture andmimicked by ionomycin in the absence of FCS. The intracellularCa²⁺ concentration([Ca²⁺]_i)during stimulation with high-K⁺solution or carbachol was reduced after culture with FCS, whereas the[Ca²⁺]_i-forcerelationship was unaffected. Cells were isolated from cultured strips,and whole cell voltage-clamp experiments were performed. Maximum inwardCa²⁺ current (10 mMBa²⁺), normalized to cellcapacitance, was almost three times smaller in cells isolated fromstrips cultured with FCS. Culture with 1 µM verapamil prevented thisreduction. These results suggest that increased[Ca²⁺]_iduring culture downregulates Ca²⁺current density, with associated effects on contractility.

     

Photosynthetic and Heterotrophic Ferredoxin Isoproteins Are Colocalized in Fruit Plastids of Tomato

Fruit tissues of tomato (Lycopersicon esculentum Mill.) contain both photosynthetic and heterotrophic ferredoxin (FdA and FdE, respectively) isoproteins, irrespective of their photosynthetic competence, but we did not previously determine whether these proteins were colocalized in the same plastids. In isolated fruit chloroplasts and chromoplasts, both FdA and FdE were detected by immunoblotting. Colocalization of FdA and FdE in the same plastids was demonstrated using double-staining immunofluorescence microscopy. We also found that FdA and FdE were colocalized in fruit chloroplasts and chloroamyloplasts irrespective of sink status of the plastid. Immunoelectron microscopy demonstrated that FdA and FdE were randomly distributed within the plastid stroma. To investigate the significance of the heterotrophic Fd in fruit plastids, Glucose 6-phosphate dehydrogenase (G6PDH) activity was measured in isolated fruit and leaf plastids. Fruit chloroplasts and chromoplasts showed much higher G6PDH activity than did leaf chloroplasts, suggesting that high G6PDH activity is linked with FdE to maintain nonphotosynthetic production of reducing power. This result suggested that, despite their morphological resemblance, fruit chloroplasts are functionally different from their leaf counterparts.     

Isolation of membranes containing protease from the cotyledons of germinating pea seeds

Membranes containing protease were isolated from the cotyledonsof germinating pea seeds by differential and two successivesucrose density gradient centrifugations. Membranes were recoveredin the microsomal fraction, but could be clearly separated frommembranes carrying antimycin A-insensitive NADH-cytochrome creductase EC 1.6.2.1 [EC] ), which seem to come from the endoplasmicreticulum. The density of protease-containing membranes was1.135. Membranes contained a lower amount of phospholipid ascompared with the endoplasmic reticulum. Electron microscopicobservations also showed that the membranes were different fromthe smooth-surfaced endoplasmic reticulum. ¹Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received September 12, 1973; )     

Envelope Permeability to Possible Precursors of Carotenoid Biosynthesis during Chloroplast-Chromoplast Transformation

During the transformation of chloroplasts to chromoplasts in Capsicum annuum L., the permeability of the envelope membranes to possible precursors of carotenoid biosynthesis (acetate, mevalonate, citrate) was tested. The plastids were isolated by gel filtration, and the uptake of labeled compounds was measured by a filtering centrifugation technique, using silicone oil. The different ripening stages were characterized by the ratio of chlorophyll to carotenoid content. The chloroplast membranes were shown to be impermeable to all of the metabolites tested. During the transformation there was a sharp increase of membrane permeability. In the mature chromoplast, the permeability to mevalonate and acetate again decreased to about 20% of the maximum value and reached zero for citrate. The results give evidence that during the transformation of chloroplasts to chromoplasts, precursors for carotenoid biosynthesis are translocated from extraplastidic sites into the plastids, there being possibly incorporated into carotenoids.     

Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue

Background and Aims

There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase.

Methods

Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices.

Key results

At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts.

Conclusions

These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.     

Molecular analysis of a phytohemagglutinin-defective cultivar of Phaseolus vulgaris L.

Plastoglobuli have been isolated and purified from chloroplasts of beech and spinach leaves and from broom flower chromoplasts by a repeated floating-gradient technique. The main components in plastoglobuli isolated from chloroplasts were triacylglycerols and lipophilic prenyl quinones, mainly plastohydroquinone and -tocopherol. The corresponding oxidized prenyl quinones, plastoquinone (ox), -tocoquinone, and the phylloquinone vitamin K₁, were detected in trace amounts. Plastoglobuli isolated from chromoplasts contained large amounts of carotenoid esters. Triacylglycerols constituted two-thirds of the content of these plastoglobuli. The total prenyl quinone content was low in chromoplast plastoglobuli. Plastoquinone (ox) was the major prenyl quinone constituent. Plastoglobuli contained small amounts of chlorophylls, carotenoids (with the exception of chromoplast plastoglobuli), glycolipids, and proteins due to adsorption phenomena during the isolation process; however, increasing purification of the plastoglobuli fractions resulted in an exponential decline of these components. Adsorption of thylakoid lipids onto the plastoglobuli during the isolation process was demonstrated using an artificial globuli system. Therefore, pigments, glyco- and phospholipids, and proteins were regarded as thylakoid contaminations and not as actual constituents of plastoglobuli.     

Structure,composition, and distribution of plastid nucleoids in Narcissus pseudonarcissus

The size, frequency and distribution of the nucleoids of chloroplasts (cl-nucleoids) and chromoplasts (cr-nucleoids) of the daffodil have been investigated in situ using the DNA-specific fluorochrome 46-diamidino-2-phenylindole. Chromoplasts contain fewer nucleoids (approx. 4) than chloroplasts (> 10), and larger chromoplasts (cultivated form, approx. 4) contain more than smaller ones (wild type, approx. 2). During chromoplast development the nucleoid number decreases in parallel with the chlorophyll content. Each nucleoid contains 2–3 plastome copies on average. In chloroplasts the nucleoids are evenly distributed, whereas they are peripherally located in chromoplasts. The fine structure of isolated cl-and cr-nucleoids, purified either by Sepharose 4B-CL columns or by metrizamide gradients, was investigated electron microscopically. The cl-nucleoids consist of a central protein-rich core with naked DNA-loops protruding from it. In cr-nucleoids, on the other hand, the total DNA is tightly packed within the proteinaceous core. The protein-containing core region of the nucleoids is made up of knotty and fibrillar sub-structures with diameters of 18 and 37 nm, respectively. After proteinase treatment, or incressing ion concentration, most of the proteins are removed and the DNA is exposed even in the case of cr-nucleoids, the stability of which proved to be greater than that of cl-nucleoids. The chemical composition of isolated plastid nucleoids has been determined qualitatively and quantitatively. Chromoplast-nucleoids contain, relative to the same DNA quantity, about six times as much protein as cl-nucleoids. Accordingly the buoyant density of cr-nucleoids in metrizamide gradients is higher than that of cl-nucleoids. In addition to DNA and protein, RNA could be found in the nucleoid fraction. No pigments were present. The cr-and cl-nucleoids have many identical proteins. There are, however, also characteristic differences in their protein pattern which are possibly related to the different expression of the genomes of chloroplasts and chromoplasts. Nucleoids of both plastid types contain some proteins which also occur in isolated envelope membranes (probably partly in the outer membrane) and thus possibly take part in binding the DNA to membranes.Abbreviations cl- chloroplast - cr- chromoplast - DAPI 46-diamidino-2-phenylindole - DNase deoxyribonuclease - kDa kilodaltons - MG purified by metrizamide gradients - SC purified by Sepharose CL-4B column gel filtration - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

An ATP synthase harboring an atypical γ–subunit is involved in ATP synthesis in tomato fruit chromoplasts

Chromoplasts are non‐photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non‐photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical γ–subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ–subunits. Silencing of this atypical γ–subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the γ–subunit present in tomato leaf and green fruit chloroplasts by the atypical γ–subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle.     

Terpenoid Metabolism in Plastids : Isolation and Biochemical Characteristics of Capsicum annuum Chromoplasts

A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl₂ in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids.     

Chromoplasts of Tropaeolum majus L.: Lipid synthesis in whole organelles and subfractions

Isolation of tubulous chromoplasts from Tropaeolum majus L. petals was achieved in pure form. Their main substructures-lipid bodies, tubules, and envelope membranes-have been enriched. Whole chromoplasts as well as substructures have been tested for their activities in lipid synthesis. The following activities were found: fatty acid synthesis from acetate, glycosyl transfer reactions from UDP-galactose and UDP-glucose to galactolipids and sterols, acyltransferase reactions from palmitoyl-CoA, and a very active acyl-CoA hydrolase (EC 3.1.2.2.). Fatty acid synthesis was restricted to whole chromoplasts. Glycosyl- and acyltransferases were essentially confined to envelope membranes, whereas acyl-CoA hydrolase was found in all fractions. The chemical composition of chromoplast subfractions was determined. The lipid bodies consisted mainly of galactolipids and carotenoid esters in a 1:1 ratio, together with small amounts of protein.     

Fatty acid synthesis by isolated chromoplasts from the daffodil. Energy sources and distribution patterns of the acids

1. Fatty acid synthesis in isolated intact chromoplasts from [1-¹⁴C]acetate was made possible by using ATP, ADP (via adenylate kinase), and, with decreasing efficiency, UTP, CTP, and GTP as energy sources. 2. The glycolytic path from dihydroxyacetone phosphate to acetyl-CoA operates within the chromoplasts. The glycolytic intermediates, especially 2-phosphoglycerate and phosphoenolpyruvate, served as very effective energy donors for fatty acid synthesis by phosphorylating the endogenous adenine nucleotide pool. 3. In the presence of exogenous ATP or ADP, appreciable amounts of in vitro formed fatty acids were found as acyl-CoA and subsequent products, mainly phosphatidylcholine. When other energy sources were used most of the acids formed were in the free form, and to a minor extent, in the phosphatidic acid and diacylglycerol fractions. Similar results have recently been reported for spinach chloroplasts (Kleinig and Liedvogel 1979, FEBS Lett.101, 339–342).Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - UTP uridine triphosphate - CTP cytidine triphosphate - GTP gnanosine triphosphate     

Protein Bodies from Buckwheat Seed Cotyledons: Isolation and Characteristics

Protein bodies were isolated from cotyledons of dry buckwheatseeds by homogenization in acetone with subsequent purificationin a 1.26 g cm^–3 to 1.53 g cm^–3 linear density gradientof a mixture of acetone with CCI₄. The purified fraction ofprotein bodies with globoids (PB I) had a buoyant density of1.48–1.51 g cm^–3 and was intact according to microscopicdata. Localization of hydrolytic enzymes and proteinase inhibitorsin the PB I fraction and in the fraction of the cytoplasm andmembrane material (CMM) was studied. It was shown that acidhydrolytic enzymes, such as aspartic proteinase, carboxypeptidase,acid phosphatase, -D-mannosidase and N-acetyl-ß-glucosaminidase,as well as chymotrypsin and trypsin inhibitors were predominantlylocalized in the PB I. BAPAase and SH-activated caseinase activitieswere equally distributed between the PB I and CMM fraction.The activities of leucine aminopeptidase and SH-independentcaseinase were noticeably predominant in the CMM fraction. Key words: Buckwheat, subcellular fractionation, protein bodies, hydrolases