Chromoplast development in a carotenoid mutant of maize

Summary The lethal recessive mutantlycopenic in maize is characterized by the synthesis of lycopene instead of the normal carotenoids. At normal conditions of illumination it loses chlorophyll by photo-oxidation. Seedlings of this mutant and of normal maize were grown at light intensities of 25–30 lux and 500–30,000 lux. Their plastid development was studied by electron microscopy.At low light intensities a kind of mesophyll chloroplast with elongated grana, long unpaired thylakoid segments, and sometimes prolamellar bodies is formed in mutant plants. In corresponding bleached plants the plastids are transformed into chromoplasts containing characteristic lycopene crystalloids similar to those found in tomato fruits. Various stages in this chromoplast development are described and illustrated. Also bundle-sheath plastids were found to develop into chromoplasts.It is concluded that the ultrastructure of plastids in a tissue is influenced by the nature of their pigments and that an altered carotenoid composition therefore can give rise to development of chromoplasts in plants which normally lack such organelles.     

Chromoplasts of Tropaeolum majus L.: Structure and development

Summary The fine structure of chromoplasts in epidermal cells of flower petals of Tropaeolum has been investigated by light, polarizing, and electron microscopy at different stages of development. The pale greenish-yellow petals still enclosed in the bud contain barely differentiated chloroplasts with few, irregular grana. The chromoplasts of unfolding petals show differently oriented bundles of tubules with variable diameters (mean: 17 nm). Thylakoid membranes become reduced more and more. The tubular bundles are intermingled with numerous isodiametric bodies of ca. 50 nm diameter; these bodies are better discernible at later stages when the chromoplasts possess a less dense matrix. The chromoplasts of open flowers are in a state of disorganization at a time when the cytoplasm still appears normal. A comparison is made between chromoplast tubules and tubular structures described from other kinds of plastids. The observations are discussed in view of chromoplast typology and with regard to possible processes underlying chromoplast differentiation in flowers.Abbreviations in Figures Chr chromoplast - CT chromoplast tubules - Cy cytoplasm - D dictyosome - IB isodiametric body - M mitochondrion - MT microtubule - oG osmiophilic globule - S S-body - St starch grain - V vacuole All micrographs from glutaraldehyde-OsO₄-fixed material, unless otherwise specified. The bar designates 1 m (multiples or fractions of it indicated).     

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O₂-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways.Plastids are developmentally related organelles capable of interconversion among a variety of structurally and biochemically distinct forms in response to both environmental and tissue-specific cues (Whatley, 1978; Thomson and Whatley, 1980). Formation of chromoplasts in many fruits is one striking example of this plasticity. Heavily pigmented, photosynthetically inactive chromoplasts frequently develop from chloroplasts during ripening. This conversion involves dramatic changes in the organization and composition of the internal plastid compartment, which include the loss of proteins involved in carbon fixation in the stroma and replacement with chromoplast-specific proteins, the breakdown of the photosynthetic thylakoid membranes and loss of proteins involved in light capture and electron transfer, and, in some cases, the formation of new membranes (Spurr and Harris, 1968; Camara and Brangeon, 1981; Piechulla et al., 1987; Kuntz et al., 1989).Chromoplast formation is an active rather than simply a degradative process. New proteins, specific to or enhanced in chromoplasts, are synthesized and compartmentalized in the plastid (Camara et al., 1995; Price et al., 1995). Most chromoplast proteins are predicted to be nuclear encoded, translated on cytoplasmic ribosomes, and posttranslationally imported into the plastid, as are nuclear-encoded chloroplast proteins. Import of chloroplast proteins occurs via a general import machinery that appears to mediate translocation of most or all proteins that are delivered to the chloroplast stroma, either as a final destination or as an intermediate location (Cline and Henry, 1996; Robinson and Mant, 1997; Schnell, 1998). Proteins are targeted to the general import pathway by an N-terminal extension that is cleaved upon import, resulting in the appearance of a processed protein of reduced M_r. Presumably, the import of proteins into chromoplasts is accomplished by the same machinery that is responsible for import of proteins into chloroplasts, although this has never been directly examined.In some chromoplasts an extensive set of internal membranes accumulates, replacing the thylakoids. For example, in the fibrillar-type chromoplast of bell pepper (Capsicum annuum), the photosynthetic membranes are replaced by membranous sheets and vesicles in addition to the carotenoid-rich plastoglobules and fibrils (Spurr and Harris, 1968; Laborde and Spurr, 1973; Camara and Brangeon, 1981; Deruere et al., 1994). The often extensive internal membranes are the site of synthesis of keto-xanthophylls, which constitute the major carotenoids of red fruit (Bouvier et al., 1994).Our interests are in the biogenesis of the internal membranes of plastids, in particular the proteins that are integral to the bilayer, as well as those located in the luminal compartment formed by the bilayer. In chloroplasts, proteins destined for the thylakoid membrane or lumen are routed from the stroma into the thylakoid membrane and lumen by one of at least four distinct mechanisms: the ΔpH, chloroplast SRP, thylakoid Sec pathways, and an apparently spontaneous insertion mechanism (for review, see Cline and Henry, 1996; Robinson and Mant, 1997; Schnell, 1998). In view of the extensive internal membrane system of bell pepper chromoplasts, one would expect the presence of proteins and accompanying translocation machinery in these membranes. However, no chromoplast-specific proteins have been conclusively demonstrated to be either integral or luminal to these membranes.One protein, Pftf (plastid fusion/translocation factor), predicted to be membrane anchored by sequence analysis, has been purified from the stromal compartment of pepper chromoplasts (Hugueney et al., 1995). This raised the possibility that mature chromoplasts lack the ability to localize proteins into/across internal membranes. To address this question we developed a method for isolating protein import-competent chromoplasts from bell peppers. Immunoblotting confirmed that these chromoplasts contain known translocation machinery components. Chromoplasts were assayed in vitro for their ability to import and localize passenger proteins from the three known protein-machinery-dependent thylakoid-targeting pathways. We found mature chromoplasts to be capable of membrane targeting of proteins that utilize the thylakoidal Sec and ΔpH pathways but not capable of inserting a membrane protein, LHCP, which utilizes the chloroplast SRP pathway. Pftf was inserted into the membranes of these chromoplasts in a manner similar to that observed in chloroplasts, and resident Pftf was also found to be integrally associated with chromoplast membranes. The precise role of these pathways in the formation of bell pepper chromoplasts remains to be fully elucidated.     

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.     

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis)

Summary. The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of -carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as membranous chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.     

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.     

Enhancement of Carotenoid Biosynthesis in Transplastomic Tomatoes by Induced Lycopene-to-Provitamin A Conversion

Carotenoids are essential pigments of the photosynthetic apparatus and an indispensable component of the human diet. In addition to being potent antioxidants, they also provide the vitamin A precursor β-carotene. In tomato (Solanum lycopersicum) fruits, carotenoids accumulate in specialized plastids, the chromoplasts. How the carotenoid biosynthetic pathway is regulated and what limits total carotenoid accumulation in fruit chromoplasts is not well understood. Here, we have introduced the lycopene β-cyclase genes from the eubacterium Erwinia herbicola and the higher plant daffodil (Narcissus pseudonarcissus) into the tomato plastid genome. While expression of the bacterial enzyme did not strongly alter carotenoid composition, expression of the plant enzyme efficiently converted lycopene, the major storage carotenoid of the tomato fruit, into provitamin A (β-carotene). In green leaves of the transplastomic tomato plants, more lycopene was channeled into the β-branch of carotenoid biosynthesis, resulting in increased accumulation of xanthophyll cycle pigments and correspondingly reduced accumulation of the α-branch xanthophyll lutein. In fruits, most of the lycopene was converted into β-carotene with provitamin A levels reaching 1 mg per g dry weight. Unexpectedly, transplastomic tomatoes also showed a >50% increase in total carotenoid accumulation, indicating that lycopene β-cyclase expression enhanced the flux through the pathway in chromoplasts. Our results provide new insights into the regulation of carotenoid biosynthesis and demonstrate the potential of plastids genome engineering for the nutritional enhancement of food crops.Carotenoids are isoprenoid molecules that are synthesized by all photosynthetic organisms and also by some fungi and nonphotosynthetic bacteria. In plants, they participate in photosynthetic light harvesting and protection against light stress. In addition, carotenoids accumulate to large levels as storage metabolites in chromoplasts of flowers, fruits, and taproots. Carotenoids are also essential to animals, which, however, are unable to synthesize them de novo, and therefore must rely on dietary sources of carotenoids. β-Carotene is the main dietary precursor of vitamin A and therefore also referred to as provitamin A. Vitamin A deficiency in humans represents a global health problem affecting approximately one-third of the countries of the world (Mayer et al., 2008). Presumably due to their antioxidant activity, β-carotene and other carotenoid species also exert protective effects against cardiovascular diseases, certain cancers, and aging-related diseases (Collins, 1999).While the enzymology of the carotenoid biosynthetic pathways in plants and eubacteria is now reasonably well understood (Armstrong, 1997; Cunningham and Gantt, 1998; Hirschberg, 2001), understanding of the regulation of carotenoid biosynthesis is still rather poor (Bramley, 2002). Mainly using the tomato (Solanum lycopersicum) fruit as model system, the study of pigmentation mutants (Ronen et al., 2000; Isaacson et al., 2002; Galpaz et al., 2006) and transgenic approaches (Giuliano et al., 2000, 2008; Römer and Fraser, 2005; Fraser et al., 2007) have provided first insights into regulatory mechanisms operating in carotenogenesis. For example, constitutive expression of the phytoene desaturase (crtI) gene from the bacterium Erwinia uredovora resulted in elevated β-carotene accumulation in tomatoes, but also led to an unexpected reduction in total carotenoid levels (Römer et al., 2000). The reduction in total carotenoids is believed to be an effect of feedback regulation from β-carotene or one of its downstream metabolites (Bramley, 2002). However, fruit-specific overexpression of the native lycopene β-cyclase resulted in increased β-carotene accumulation, without a concomitant decrease in total carotenoids (Rosati et al., 2000). Why some genetic disturbances of carotenoid biosynthesis negatively affect total carotenoid accumulation and others do not (or even result in an increase; Dharmapuri et al., 2002; Fraser et al., 2002), remains to be established.Here we have used tomato plastid transformation to address the regulation of carotenoid biosynthesis exerted at the level of lycopene to β-carotene conversion by the enzyme lycopene β-cyclase (Fig. 1A). We show that plastid expression of a plant lycopene β-cyclase does not only trigger efficient conversion of lycopene to β-carotene, but unexpectedly also results in a >50% increase in total carotenoid accumulation. This contrasts moderately increased β-carotene levels and reduced total carotenoid accumulation upon expression of a bacterial lycopene β-cyclase (Wurbs et al., 2007) and suggests lycopene β-cyclase activity as an important regulatory point in plant and microbial carotenoid biosynthesis.Open in a separate windowFigure 1.Engineering of the carotenoid biosynthetic pathway by plastid transformation. A, Carotenoid biosynthetic pathway in higher plants. The pathway splits into an α-branch and a β-branch immediately downstream of lycopene, the major storage carotenoid of tomato fruits. The enzyme expressed from the tomato plastid genome in this study, lycopene β-cyclase, leads into the β-branch. B, Physical maps of the targeting region in the plastid genome (ptDNA) and the plastid transformation vectors pEcrtY and pNLyc constructed in this study. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic region between the trnfM and trnfG genes (Ruf et al., 2001). The selectable marker gene aadA is driven by a chimeric rRNA operon promoter (Prrn; Svab and Maliga, 1993), fused to the 3′-UTR from the psbA gene (TpsbA), and flanked by two loxP sites to allow marker removal by Cre-mediated site-specific recombination (Zhou et al., 2008). The transgene expression cassette consists of the ribosomal RNA operon promoter fused to the 5′ leader from the gene 10 of phage T7 (Prrn-G10L; Kuroda and Maliga, 2001) and the 3′-UTR of the rps16 gene (Trps16). Restriction sites used for cloning or RFLP analysis are indicated, and the psaB-derived hybridization probe is denoted by a horizontal bar. Sites lost due to ligation to heterologous ends are in parentheses. C, Southern-blot analysis of tomato transplastomic lines carrying the lycopene β-cyclase gene from daffodil (S.l.-pNLyc) or from E. herbicola (S.l.-pEcrtY). Total cellular DNA was digested with BglII and hybridized to a radioactively labeled probe detecting the psaB region of the plastid genome, which flanks the transgene insertion site (section B). Fragment sizes are given in kb. wt, Wild type. D, Alignment (produced with ClustalW2) of the amino acid sequences of the lycopin β-cyclases from daffodil (Np) and E. herbicola (Eh). Asterisk (*) denotes residues identical in both sequences (marked in bold), colon (:) indicates conserved substitutions, and a dot indicates semiconserved substitutions. The N-terminal extension of the Np sequence is likely to harbor the transit peptide for protein import into plastids. The amino acids that changed due to correction of the Lyc sequence from daffodil (published sequence: GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"X98796.1","term_id":"1419692"}}X98796.1; corrected sequence: accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ327929","term_id":"254681605"}}GQ327929) are underlined. The corrections improve the sequence similarity in the N-terminal domains of the Np and Eh sequences.     

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.     

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.     

Chromoplast structures inThunbergia flowers

Summary The differentiation of tubulous chromoplasts in developing flowers ofThunbergia alata was studied by ultrastructural, pigment and protein analyses. The way of chromoplast formation in the mesophyll differed from that in the adaxial epidermis. While, in mesophyll cells, the chloroplasts were directly transformed into chromoplasts of the tubulous type, characteristic membranes and the tubular reticulum appeared in the adaxial epidermal cells, before the formation of tubules. The disappearance of the photosynthetic apparatus, the formation of membranous structures and the appearance of tubules were studied. The tubulous chromoplasts contained a 32 kDa protein, an unidentified carotene, and small quantities of lutein and -carotene.Abbreviations DAB diaminobenzidine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Phytochrome-mediated Carotenoid Biosynthesis and Its Influence by Plant Hormones

Lycopenc biosynthesis of parenchyma chromoplasts was studied in detached tomato fruits, Lycopersicum esculentum Mill, cv. Waltham Forcing, and found to be phytochrome mediated. A few minutes of red light during the day enhanced lycopene formation. Far-red irradiation did not enhance lyco-pene biosynthesis. Far-red following red nullified the promotive effect of red light. Lycopene content increased two-fold in the presence of abscisic acid. Ripening of tomatoes was inhibited when gibberellin, kinetin and ascorbic acid were applied to green tomatoes. Gibberellin (A₃) was more inhibitory to lycopene synthesis than kinetin.     

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     

Gene expression and biological pathways in tissue of men with prostate cancer in a randomized clinical trial of lycopene and fish oil supplementation

Background

Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial.

Methods

Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients.

Results

Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001).

Conclusions

We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00402285","term_id":"NCT00402285"}}NCT00402285     

A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh

Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast biogenesis, differentiation, and senescence in sweet orange flesh.Chromoplasts are special organelles with superior ability to synthesize and store massive amounts of carotenoids, bringing vivid red, orange, and yellow colors to many flowers, fruits, and vegetables (Li and Yuan, 2013). Chromoplasts exhibit various morphologies, such as crystalline, globular, tubular, and membranous structures (Egea et al., 2010). The relationship between the architecture and carotenoid composition has been well stated in diverse pepper (Capsicum annuum) and tomato (Solanum lycopersicum) fruits (Kilcrease et al., 2013; Nogueira et al., 2013). Crystalline bodies have been observed in carrot (Daucus carota; Frey-Wyssling and Schwegler, 1965) and tomato (Harris and Spurr, 1969), which predominantly consist of β-carotene and lycopene, respectively. Globular and/or tubular-globular chromoplasts, in which numerous lipid droplets (also named plastoglobules), which act as passive storage compartments for triglycerides, sterol ester, and some pigments, are accumulated, were described for yellow fruits from kiwi (Actinidia deliciosa), papaya (Carica papaya), and mango (Mangifera indica), which contain lutein, β-cryptoxanthin, and β-carotene as the major pigments, respectively (Vasquez-Caicedo et al., 2006; Montefiori et al., 2009; Schweiggert et al., 2011). Carotenoid composition has been reported to be regulated by the expression of carotenogenic genes in the flesh of various citrus fruits differing in their internal colors (Fanciullino et al., 2006, 2008). Chromoplasts are frequently derived from fully developed chloroplasts, as seen during fruit ripening from green to red or yellow fruits in tomato and pepper (Egea et al., 2010). In some cases, chromoplasts also arise from nonphotosynthetic plastids, such as colorless proplastids, leucoplasts, or amyloplasts (Knoth et al., 1986; Schweiggert et al., 2011). To date, most studies on chromoplast differentiation have been focused on the synthesis of carotenoids by combining biochemical and molecular analyses (Cazzonelli and Pogson, 2010; Egea et al., 2010; Bian et al., 2011; Li and Yuan, 2013), and little is known about the molecular mechanisms underlying chromoplast biogenesis (Li and Yuan, 2013).Recently, proteomics has become an efficient tool to study the protein composition of subcellular organelles such as chromoplasts and their dynamic changes during the development of a particular plant organ/tissue. The majority of chromoplast-related studies are concerned with the functions of these organelles in various crops, such as pepper, tomato, watermelon (Citrulis lanatus), carrot, cauliflower (Brassica oleracea), and papaya (Siddique et al., 2006; Wang et al., 2013). However, only a few of such studies addressed the mechanisms underlying plastid differentiation, such as the transition from proplastid to chloroplast in maize (Zea mays; Majeran et al., 2010), from etioplast to chloroplast in pea (Pisum sativum; Kanervo et al., 2008) and rice (Oryza sativa; Kleffmann et al., 2007), and from chloroplast to chromoplast in tomato (Barsan et al., 2012). In tomato, chromoplastogenesis appears to be associated with major metabolic shifts, including a strong decrease in abundance of the proteins involved in light reaction and an increase in terpenoid biosynthesis and stress-response proteins (Barsan et al., 2012). These changes in proteins are in agreement with the structural changes occurring in tomato during fruit ripening, which is characterized by the loss of chlorophyll and the synthesis of colored compounds. Chromoplast differentiation from nonphotosynthetic plastids occurs frequently in a number of plant tissues, such as watermelon flesh and carrot root (Kim et al., 2010; Wang et al., 2013). However, to the best of our knowledge, no large-scale proteomic study for understanding this developmental process has been reported.Citrus is one of the most economically important fruit crops in the world. Different from the model fruit tomato, which represents climacteric fruits, citrus shows nonclimacteric characteristics during fruit maturation. Additionally, citrus fruits exhibit a unique anatomical fruit structure consisting of two major sections, the pericarp and the edible flesh. Considerable progress has been made in the understanding of chromoplast differentiation in the pericarp of citrus fruits (Eilati et al., 1969; Iglesias et al., 2007), which is a process similar to that of tomato and pepper (Egea et al., 2010). However, little is known about the molecular basis of chromoplast differentiation in the edible flesh, even though there is increasing evidence suggesting an essential role of carotenoid synthesis in inducing chromoplast differentiation (Egea et al., 2010; Bian et al., 2011; Li and Yuan, 2013). Recently, we successfully isolated and purified intact chromoplasts containing a large number of plastoglobules from the flesh of sweet orange (Citrus sinensis) fruits at the maturation stage (Zeng et al., 2011). The same method has also been used successfully to isolate plastids from sweet orange flesh in earlier maturation stages (Zeng et al., 2014), thus making comparative and quantitative proteomic analyses of plastid differentiation possible. In this study, we investigated how ultrastructural changes of plastids/chromoplasts during sweet orange fruit maturation might be associated with changes in the composition of carotenoids and the expression of carotenogenic genes in red and yellow flesh of the fruits. Furthermore, we employed the isobaric tag for relative and absolute quantitation (iTRAQ)-based technology to investigate how protein compositional changes might be correlated with metabolic and structural changes in the plastids of sweet orange flesh during their transformation from amyloplasts to chromoplasts.     

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.     

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.     

Biorefinery cascade processing for creating added value on tomato industrial by-products from Tunisia

Background

In today’s consumer perception of industrial processes and food production, aspects like food quality, human health, environmental safety, and energy security have become the keywords. Therefore, much effort has been extended toward adding value to biowastes of agri-food industries through biorefinery processing approaches. This study focused, for the first time, on the valorization of tomato by-products of a Tunisian industry for the recovery of value-added compounds using biorefinery cascade processing.

Results

The process integrated supercritical CO₂ extraction of carotenoids within the oil fractions from tomato seeds (TS) and tomato peels (TP), followed by a batch isolation of protein from the residues. The remaining lignocellulosic matter from both fractions was then submitted to a liquid hot water (LHW) hydrolysis. Supercritical CO₂ experiments extracted 5.79% oleoresin, 410.53 mg lycopene/kg, and 31.38 mg β-carotene/kg from TP and 26.29% oil, 27.84 mg lycopene/kg, and 5.25 mg β-carotene/kg from TS, on dry weights. Protein extraction yields, nearing 30% of the initial protein contents equal to 13.28% in TP and 39.26% in TS, revealed that TP and TS are a rich source of essential amino acids. LHW treatment run at 120–200 °C, 50 bar for 30 min showed that a temperature of 160 °C was the most convenient for cellulose and hemicellulose hydrolysis from TP and TS, while keeping the degradation products low.

Conclusions

Results indicated that tomato by-products are not only a green source of lycopene-rich oleoresin and tomato seed oil (TSO) and of protein with good nutritional quality but also a source of lignocellulosic matter with potential for bioethanol production. This study would provide an important reference for the concept and the feasibility of the cascade fractionation of valuable compounds from tomato industrial by-products.

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Graphical abstract Schema of biorefinery cascade processing of tomato industrial by-products toward isolation of valuable fractions.