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.     

Tomato Fruit Chromoplasts Behave as Respiratory Bioenergetic Organelles during Ripening

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b₆f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c₆ during ripening suggests that it could act as electron acceptor of the cytochrome b₆f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages.Chromoplasts are plastids specialized in the production and accumulation of carotenoids, conferring color to many fruits and flowers. During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into chromoplasts in a process that involves the dismantling of the photosynthetic apparatus and a massive synthesis and deposition of lycopene (Camara et al., 1995). Chromoplasts show a barely studied respiratory process, first reported for daffodil (Narcissus pseudonarcissus) chromoplasts and called chromorespiration, which consists of a membrane-bound redox pathway associated with carotenoid desaturation and results in oxygen uptake activity (Nievelstein et al., 1995). The most likely oxidase involved in this respiratory activity is the plastidial terminal oxidase (PTOX), a plastoquinol oxidase homologous to the mitochondrial alternative oxidase (AOX; Carol et al., 1999; Wu et al., 1999). According to its role in chromorespiration and in carotenoid biosynthesis, the expression of PTOX increases during the ripening process of tomato and bell pepper (Capsicum annuum) fruits (Josse et al., 2003), in parallel to chromoplast differentiation. PTOX has been characterized in vitro and it has been reported to be inhibited by pyrogallol analogs, specially by octyl gallate (Ogal; Josse et al., 2000). In vivo, PTOX has been studied mainly in chloroplasts. PTOX not only participates in carotenoid biosynthesis in chloroplasts but is also involved in chlororespiration, an electron transport chain present in thylakoids that shares plastoquinone with the photosynthetic electron transport chain (Carol and Kuntz, 2001; McDonald et al., 2011).In daffodil chromoplast homogenates (Nievelstein et al., 1995) as well as in isolated tomato fruit chromoplasts (Pateraki et al., 2013), NAD(P)H acts as an electron donor for chromorespiration, indicating the participation of NAD(P)H plastoquinone oxidoreductase activity. Considering that tomato fruit chromoplasts derive from chloroplasts, it is possible that some components of the chromoplastic redox pathway could originate from chlororespiration, such as the NAD(P)H:plastoquinone-reductase complex (NDH), which could act as the electron entrance. However, the enzymes involved in chromorespiration are not well known. It was also reported that the oxygen uptake activity of daffodil chromoplast homogenates was sensitive to the classic uncoupler 2,4-dinitrophenol (Nievelstein et al., 1995), and this observation led to the proposal that chromorespiration could be linked to membrane energization. Morstadt et al. (2002) found that liposomes containing daffodil chromoplast proteins and energized by an acid-base transition were able to produce ATP through a chemiosmotic mechanism, demonstrating that daffodil chromoplasts contain a functional H⁺-ATP synthase complex. We recently reported that isolated chromoplasts from tomato fruits can synthesize ATP de novo (Pateraki et al., 2013). This process is dependent on an ATP synthase complex containing an atypical γ-subunit without the regulatory dithiol domain, which may be active using lower proton gradients than those present in the chloroplast (Pateraki et al., 2013). This finding is consistent with proteomic analyses that reveal that several proteins related to electron transport and ATP production are present in chromoplasts of ripe fruits, like ATP synthase, some subunits of the NDH complex, and the cytochrome b₆f complex (Barsan et al., 2012; Wang et al., 2013).Several anabolic pathways that require ATP and reducing agents are active in ripe fruit chromoplasts, such as synthesis of carotenoids, lipids (glycolipids, phospholipids, and sterols), and the shikimate pathway (Bian et al., 2011; Angaman et al., 2012). On the other hand, the ATP synthesis capacity of mitochondria in ripe fruit is low, because its membrane potential diminishes during ripening as a result of the increasing activity of the mitochondrial uncoupling protein (Almeida et al., 1999; Costa et al., 1999). This fact raised the question of whether chromorespiration could play a significant role in the production of ATP at the last stages of ripening. To our knowledge, the ATP synthesis rates of chromoplasts have not been quantified; therefore, it was uncertain whether the endogenous production could provide ATP in significant amounts to address the energy requirements of the chromoplasts. Moreover, there was no information about the quantitative contribution of chromorespiration to total fruit tissue respiration. This work aimed to deepen the study of the chromorespiratory process in isolated tomato fruit chromoplasts and to analyze the relative participation of this pathway in the overall respiration and ATP levels of fruit pericarp in vivo.     

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.     

Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell

Stromules are motile extensions of the plastid envelope membrane, whose roles are not fully understood. They are present on all plastid types but are more common and extensive on non-green plastids that are sparsely distributed within the cell. During tomato fruit ripening, chloroplasts in the mesocarp tissue differentiate into chromoplasts and undergo major shifts in morphology. In order to understand what factors regulate stromule formation, we analysed stromule biogenesis in tobacco hypocotyls and in two distinct plastid populations in tomato mesocarp. We show that increases in stromule length and frequency are correlated with chromoplast differentiation, but only in one plastid population where the plastids are larger and less numerous. We used tobacco hypocotyls to confirm that stromule length increases as plastids become further apart, suggesting that stromules optimize the plastid-cytoplasm contact area. Furthermore, we demonstrate that ectopic chloroplast components decrease stromule formation on tomato fruit chromoplasts, whereas preventing chloroplast development leads to increased numbers of stromules. Inhibition of fruit ripening has a dramatic impact on plastid and stromule morphology, underlining that plastid differentiation status, and not cell type, is a significant factor in determining the extent of plastid stromules. By modifying the plastid surface area, we propose that stromules enhance the specific metabolic activities of plastids.     

珊瑚豆果实成熟过程中叶绿体转化为杂色体的研究

珊瑚豆 (Solanum pseudo- capsicum var.diflorum (Vell.) Bitter)果实成熟过程中 ,果实颜色的变化和叶绿素含量降低及类胡萝卜素含量增长相符合。对果实中叶绿体转化为杂色体进行了电镜观察。早期绿色果实的特点是叶绿体具典型的基粒 -基粒间类囊体结构。在黄绿色果实时期叶绿体类囊体系统解体 ,代之以少数非叶绿素的单个类囊体和积累大的嗜锇的质体小球。质体转变为所谓的原质体。这表明叶绿体在果实成熟中的脱分化过程。当果实达到黄色阶段 ,这些质体所含的质体小球开始从中央形成质体小管的结构。最初质体小球中央变为半透明 ,认为是质体累积胡萝卜素的开始。随着质体小球的延长 ,小管从小球中伸出。这些小管围以电子致密的膜 ,中央是半透明的轴心。与此同时 ,在质体基质中出现一系列发育不同阶段的小泡 ,似乎是形成新的质体小球的过程。在成熟的橙色和橙红色果实中的杂色体中只包含无数小管和小的质体小球。质体小管在数量和长度上增长 ,充满成熟的杂色体。无数质体小球分布在小管之间的空间中。成熟杂色体从脱分化的原质体的重建是真正的再分化过程。可以作出结论 ,珊瑚豆果实叶绿体转化为杂色体实质上是一个脱分化和再分化过程     

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.     

Studies on the Conversion of Chloroplast to Chromoplast During Fruit Ripening in Solanum pseudo-capsicum var. diflorum

Determination of chlorophyll and carotenoid contents in the ectocarp during fruit ripening in Solanum pseudo-capsicum var. diflorurn (Veil.) Bitter revealed that the changes of fruit colour coincided with the decline of chlorophyll and the increase of carotenoid contents. The conversion of chloroplasts to chromoplasts in the fruit was studied by electron microscopy. The early green fruit was characterized by chloroplasts with a typical grana-intergranal thylakoid structure. At yellow-green fruit stage the thylakoid system was disintegrated and replaced by few non-chlorophyllous single thylakoids, with accumulation of large osmiophilic plastoglobules. The plastids developed as the so-called proplastids. These indicated dedifferentiation of chloroplasts in a ripening fruit. When the fruit reached its yellow stage, numerous large plastoglobules contained in the young chromoplasts frequently showed transitional changes to plastid tubule structure. At first, the center of plastoglobules became semi-translucent. It was believed that the young chromoplast were in an initial state of carotenoid deposition, followed by plastoglobules elongation and tubule protrution from the globules. These tubules were surrounded with an electron dense membranous sheath leaving the core semi-translucent. Concurrently a series of vesicles in different developmental stages appeared from the stroma of the plastid, likely representing a process of formation of numerous small new plastoglobules. In the chromoplasts of a ripe orange-or orange red-colored fruit only numerous tubules and small plastoglobules were present. The plastid tubules increased in number and elongated in length filling the mature chromoplast. Numerous small plastoglobules also increased and distributed in the spaces between tubules. These results indicated that the reconstruction of a mature chromoplast from a dedifferentiated plastid was really a form of redifferentiation, and it might be concluded that the conversion of chloroplast to chromoplast in the fruit of S. pseudo-capsicum var. diflorum, in fact, was a processes of dedifferentiation and redifferentiation.     

Chromoplast biogenesis and carotenoid accumulation

Chromoplasts are special organelles that possess superior ability to synthesize and store massive amounts of carotenoids. They are responsible for the distinctive colors found in fruits, flowers, and roots. Chromoplasts exhibit various morphologies and are derived from either pre-existing chloroplasts or other non-photosynthetic plastids such as proplastids, leucoplasts or amyloplasts. While little is known about the molecular mechanisms underlying chromoplast biogenesis, research progress along with proteomics study of chromoplast proteomes signifies various processes and factors important for chromoplast differentiation and development. Chromoplasts act as a metabolic sink that enables great biosynthesis and high storage capacity of carotenoids. The formation of chromoplasts enhances carotenoid metabolic sink strength and controls carotenoid accumulation in plants. The objective of this review is to provide an integrated view on our understanding of chromoplast biogenesis and carotenoid accumulation in plants.     

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.     

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.     

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g(-1) and 13 μg g(-1) fresh weight (FW), respectively, and for BS 19 μg g(-1) and 0.27 μg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.     

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.     

Plastid and stromule morphogenesis in tomato

By using green fluorescent protein targeted to the plastid organelle in tomato (Lycopersicon esculentum Mill.), the morphology of plastids and their associated stromules in epidermal cells and trichomes from stems and petioles and in the chromoplasts of pericarp cells in the tomato fruit has been revealed. A novel characteristic of tomato stromules is the presence of extensive bead-like structures along the stromules that are often observed as free vesicles, distinct from and apparently unconnected to the plastid body. Interconnections between the red pigmented chromoplast bodies are common in fruit pericarp cells suggesting that chromoplasts could form a complex network in this cell type. The potential implications for carotenoid biosynthesis in tomato fruit and for vesicles originating from beaded stromules as a secretory mechanism for plastids in glandular trichomes of tomato is discussed.