Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to β-carotene when expressed in Escherichia coli . The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene β-cyclase activity when expressed in E. coli . A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re-channeling of linear carotenoids into β-cyclic carotenoids in C. annuum ripening fruits.     

Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.     

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.     

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.     

Immunocytochemical detection of ribulose bisphosphate carboxylase in capsicum plastids

Ribulose bisphosphate carboxylase (RUBPCase) was localized by fluorescence and gold immunocytochemistry in Capsicum fruits. Chloroplasts of the green fruit are heavily labelled. A positive staining is also obtained with chromoplasts of the ripe rad fruit, but gold labelling is fainter. The presence of reactive RuBPCase in chromoplasts is discussed in relation with the absence of ribosomes in these plastids.     

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.     

A putative plastidic glucose translocator is expressed in heterotrophic tissues that do not contain starch, during olive (Olea europea L.) fruit ripening

Metabolite-specific transporters are present in the inner membrane of the plastid envelope allowing transport between the plastid and other cellular compartments. A plastidic glucose translocator (pGlcT) in leaf mesophyll cells transports glucose from chloroplast stroma to the cytosol after amylolytic starch degradation at night. Here we report the cloning of a pGlcT expressed in olive fruits (Olea europea L.). Our results showed high expression of pGlcT in non-green heterotrophic fruit tissues. Expression of pGlcT in olive fruits was somewhat higher compared to leaves, and continued until the black, mature fruit stage. We cloned part of tomato pGlcT and found that it is also expressed throughout fruit development implying a role for pGlcT in heterotrophic tissues. Light and electron microscopic characterization of plastid structural changes during olive fruit ripening revealed the transition of chloroplast-like plastids into starchless, non-green plastids; in mature olive fruits only chromoplasts were present. Together, these findings suggest that olive pGlcT is abundant in chromoplasts during structural changes, and provide evidence that pGlcT may play different physiological roles in ripening fruits and possibly in other non-photosynthetic organs.     

CHROMOPLASTS OF TOMATO FRUITS. I. ULTRASTRUCTURE OF LOW-PIGMENT AND HIGH-BETA MUTANTS. CAROTENE ANALYSES

Three pigment lines of the tomato cultivar ‘Pearson’ with isogenic backgrounds were studied to determine the relationship between certain carotenoids and the development of chromoplasts during fruit ripening. The lines were normal red (r⁺/r⁺), in which about 90% of the carotenoids in the ripe fruit is lycopene; high-beta (B/B) mutant, in which beta-carotene is the major pigment and the mature fruit color is deep orange ; and low-pigment (r/r) mutant, in which carotenoids are drastically reduced and the mature fruit is pale yellow-orange. This paper reports pigment analyses for the three lines and the ultrastructural changes in plastids of the two mutant lines. Very young, pale green fruits contain proplastids with limited lamellar structure. As the fruits reach the mature green stage, the plastids in all three lines develop into typical chloroplasts. Differences in pigment content and in ultrastructure among the lines are not apparent until ripening commences. In the low-pigment mutant carotenoids are reduced as ripening progresses and no carotenoid crystalloids are formed. As chlorophyll decreases the fruits become pale yellow. The grana become disorganized and the thylakoids appear to separate at the partitions and tend to be arrayed in lines, some still with their ends overlapping. Globules increase slightly in number. In the high-beta mutant the grana break down during ripening and globules increase greatly in size and number. Beta-carotene, presumed to be largely in the globules, crystallizes into elongated or druse type forms which may distort the globules. The crystals may affect the shape of the chromoplasts; long crystals may extend the length of the plastid to over 15 μ. Thylakoid plexes with a regular lattice structure sometimes occur in the chromoplasts of the high-beta mutant. Granules resembling aggregations of phytoferritin particles occur in the chromoplasts of both of these mutants.     

Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts

We report a comprehensive proteome analysis of chromoplasts from bell pepper (Capsicum annuum L.). The combination of a novel strategy for database-independent detection of proteins from tandem mass spectrometry (MS/MS) data with standard database searches allowed us to identify 151 proteins with a high level of confidence. These include several well-known plastid proteins but also novel proteins that were not previously reported from other plastid proteome studies. The majority of the identified proteins are active in plastid carbohydrate and amino acid metabolism. Among the most abundant individual proteins are capsanthin/capsorubin synthase and fibrillin, which are involved in the synthesis and storage of carotenoids that accumulate to high levels in chromoplasts. The relative abundances of the identified chromoplast proteins differ remarkably compared with their abundances in other plastid types, suggesting a chromoplast-specific metabolic network. Our results provide an overview of the major metabolic pathways active in chromoplasts and extend existing knowledge about prevalent metabolic activities of different plastid types.     

Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase

Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-specific isoform of phytoene synthase) in wild-type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.     

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     

Ultrastructural development of plastids in cherry peppers during fruit ripening

The fine structure of plastids in the fruit of cherry peppers was studied during the various stages of ripening. The color change of fruit during ripening is due to the quantitative change of such pigment components as chlorophyll, carotenoid and anthocyanin. Plastid metamorphosis takes place in relationship to the disappearance of chlorophyll and the new formation of carotenoids. The membrane system of plastids degenerates through ripening, although a little differentiation is observed in young plastids of creamy fruits. In parallel wity the color change of fruit from cream to orange, the osmiophilic globules increase in both number and size. As ripening proceeds further, the large osmiophilic globules seem to be gradually transformed into the needle shaped crystalloids of carotenoid pigments which are the remarkable feature of the chromoplasts in red-ripe fruit. The relationship between the development of chromoplasts and the increase and decrease of some pigments is also discussed.     

Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in Capsicum chromoplasts.

In plants, zeta-carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two-step desaturation reaction: phytoene-->phytofluene--> zeta-carotene. Using Capsicum annuum chromoplast membranes and the reconstitution system previously described [Camara, B., Bardat, F. & Monéger, R. (1982) Eur. J. Biochem. 127, 255-258], we have attempted to purify the desaturase(s) catalyzing these reactions. The two activities were coincidental during all the purification procedures. Only a single polypeptide with 56 +/- 2 kDa was detected by SDS/PAGE of all active fractions. The enzyme contained protein-bound FAD. Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein. The antibodies were used to isolate a full-length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide-binding site. Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase. The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening. However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C. annuum fruits.     

Characterization of chromoplasts and carotenoids of red- and yellow-fleshed papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.)

Chromoplast morphology and ultrastructure of red- and yellow-fleshed papaya (Carica papaya L.) were investigated by light and transmission electron microscopy. Carotenoid analyses by LC–MS revealed striking similarity of nutritionally relevant carotenoid profiles in both the red and yellow varieties. However, while yellow fruits contained only trace amounts of lycopene, the latter was found to be predominant in red papaya (51% of total carotenoids). Comparison of the pigment-loaded chromoplast ultrastructures disclosed tubular plastids to be abundant in yellow papaya, whereas larger crystalloid substructures characterized most frequent red papaya chromoplasts. Exclusively existent in red papaya, such crystalloid structures were associated with lycopene accumulation. Non-globular carotenoid deposition was derived from simple solubility calculations based on carotenoid and lipid contents of the differently colored fruit pulps. Since the physical state of carotenoid deposition may be decisive regarding their bioavailability, chromoplasts from lycopene-rich tomato fruit (Lycopersicon esculentum L.) were also assessed and compared to red papaya. Besides interesting analogies, various distinctions were ascertained resulting in the prediction of enhanced lycopene bioavailability from red papaya. In addition, the developmental pathway of red papaya chromoplasts was investigated during fruit ripening and carotenogenesis. In the early maturation stage of white-fleshed papaya, undifferentiated proplastids and globular plastids were predominant, corresponding to incipient carotenoid biosynthesis. Since intermediate plastids, e.g., amyloplasts or chloroplasts, were absent, chromoplasts are likely to emerge directly from proplastids.