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.     

Stromules: a characteristic cell-specific feature of plastid morphology

Stromules (stroma-filled tubules) are highly dynamic structures extending from the surface of all plastid types examined so far, including proplastids, chloroplasts, etioplasts, leucoplasts, amyloplasts, and chromoplasts. Stromules are usually 0.35-0.85 microm in diameter and of variable length, from short beak-like projections to linear or branched structures up to 220 mum long. They are enclosed by the inner and outer plastid envelope membranes and enable the transfer of molecules as large as Rubisco (approximately 560 kDa) between interconnected plastids. Stromules occur in all cell types, but stromule morphology and the proportion of plastids with stromules vary from tissue to tissue and at different stages of plant development. In general, stromules are more abundant in tissues containing non-green plastids, and in cells containing smaller plastids. The primary function of stromules is still unresolved, although the presence of stromules markedly increases the plastid surface area, potentially increasing transport to and from the cytosol. Other functions of stromules, such as transfer of macromolecules between plastids and starch granule formation in cereal endosperm, may be restricted to particular tissues and cell types.     

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.     

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.     

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.     

Changes in the protein and activity levels of the plastid NADH–plastoquinone–oxidoreductase complex during fruit development

Plastids contain an NADH dehydrogenase complex (Ndh complex) homologous to the mitochondrial complex I (EC 1.6.5.3). In this work, we have analysed the changes in the Ndh complex during ripening of pepper (Capsicum annum L., cv. Maor) and tomato (Lycopersicon esculentum Mill., cv. Marglobe) fruits. The Ndh complex was mainly present in the outer pericarp of tomato fruits, whereas it was evenly distributed in the pericarp of pepper. In both kinds of fruit we observed a decrease in the total amount of Ndh complex from the green to the red stage of development. This decrease corresponds to parallel decreases in the content and activity of the complex in plastids during the transition from chloroplasts to chromoplasts. Levels of plastidial quinol peroxidase activity were also higher during the first stages of tomato fruit development than during the latter stages of ripening. However, when referred to total plastid protein, the amount and activity of the Ndh complex in chloroplasts isolated from green fruits was higher than in chloroplasts isolated from leaves. These results strongly suggest that function of the Ndh complex, probably related to a plastidial electron transport chain, can be important during the first stages of fruit development.     

Plastid stromules: video microscopy of their outgrowth, retraction, tensioning, anchoring, branching, bridging, and tip-shedding

Summary. Stromules are stroma-containing tubules which can grow from the surface of plastids, most commonly leucoplasts and chromoplasts, but also chloroplasts in some tissues. Their functions are obscure. Stills from video rate movies are presented here. They illustrate interaction of stromules with cytoskeletal strands and the anchoring of stromules to unidentified components at the cell surface. Anchoring leads to stretching and relaxation of stromules when forces arising from cytoplasmic streaming act on the attached, freely suspended plastid bodies. Data on stromule growth, retraction, and regrowth rates are provided. Formation and movement of stromular branches and bridges between plastids are described. The shedding of a tip region into the streaming cytoplasm is recorded in frame-by-frame detail, in accord with early observations. Correspondence and reprints: Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT 2601, Australia.     

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.     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.     

ADP-glucose pyrophosphorylase is located in the plastid in developing tomato fruit

The subcellular location of activity and protein of ADP-glucose pyrophosphorylase (AGPase) in developing tomato (Lycopersicon esculentum) fruit was determined following a report that the enzyme might be present inside and outside the plastids in this organ. Plastids prepared from crude homogenates of columella and pericarp, the starch-accumulating tissues of developing fruit, contained 8% to 18% of the total activity of enzymes known to be confined to plastids, and 0.2% to 0.5% of the total activity of enzymes known to be confined to the cytosol. The proportion of the total activity of AGPase in the plastids was the same as that of the enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antiserum raised to AGPase, most or all of the protein detected was plastidial. Taken as a whole, these data provide strong evidence that AGPase is confined to the plastids in developing tomato fruit.     

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.     

New insights on stromules: Stroma filled tubules extended by independent plastids

The recognition of stromules as sporadically extended stroma filled tubules from all kinds of plastids constitutes one of the major insights that resulted from the direct application of green fluorescent protein aided imaging of living plant cells. Observations of dynamic green fluorescent stromules strongly suggested that plastids frequently interact with each other while photo-bleaching of interconnected plastids indicated that proteins can move within the stroma filled tubules. These observations readily fit into the prevailing concept of the endosymbiogenic origins of plastids and provided stromules the status of conduits for inter-plastid communication and macromolecule transfer. However, experimental evidence obtained recently through the use of photoconvertible protein labeled stromules strongly supports plastid independence rather than their interconnectivity. Additional information on stress conditions inducing stromules and observations on their alignment with other organelles suggests that the major role of stromules is to increase the interactive surface of a plastid with the rest of the cytoplasm.     

Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis

Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all.     

Plastids and stromules interact with the nucleus and cell membrane in vascular plants

The various metabolic activities of plastids require continuous exchange of reactants and products with other organelles of the plant cell. Physical interactions between plastids and other organelles might therefore enhance the efficiency of plant metabolism. We have observed a close apposition of plastids and nuclei in various organs of Nicotiana tabacum and Arabidopsis thaliana. In hypocotyl epidermal cells, plastids and stromules, stroma-filled tubular extensions of the plastid envelope membrane, were observed to reside in grooves and infoldings of the nuclear envelope, indicating a high level of contact between the two organelle membranes. In a number of non-green tissues, including suspension-cultured cells, perinuclear plastids were frequently associated with long stromules that extended from the cell center to the cell membrane. In cotyledon petioles, cells lying adjacent to one another frequently contained stromules that met on either side of the shared cell wall, suggesting a means of intercellular communication. Our results therefore suggest that stromules have diverse roles within plant cells, perhaps serving as pathways between nuclei and more distant regions of the cell and possibly even other cells.     

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.