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.     

Synthesis and organization of vitellogenin and vitellin molecules from the land crab Potamon potamios

We have previously reported that vitellogenin (Vg) of some female animals contained four polypeptides with molecular mass of 181, 115, 105 and 85 kDa, whereas Vg of most animals contained three polypeptides with molecular mass of 115, 105 and 85 kDa. In the present investigation, we examined whether the 181 kDa polypeptide is the precursor of 115 and 105 kDa Vg and vitellin (Vn) polypeptides. Labeling studies, using [35S]methionine on normal vitellogenic animals, showed that the radioactivity was distributed first among the 181 and 85 kDa polypeptides. SDS-PAGE analysis of purified hemolymph Vg from eyestalk ablated female animals revealed in most animals two polypeptides with an apparent molecular mass of 181 and 85 kDa. These results from in vivo experiments corroborated the view that the 115 and 105 kDa Vg and Vn polypeptides are derived from heaviest 181 kDa polypeptide. In addition it was demonstrated that hepatopancreas and ovary of Potamon potamios incubated in vitro with [35S]methionine synthesized five polypeptides with apparent molecular mass of 224, 181, 115, 105, and 85 kDa while the hepatopancreas appeared to secrete the 181, 115, 105 and 85 kDa polypeptides. The major 115, 105 and 85 kDa polypeptides were found to be components of egg Vn, while the 224 kDa polypeptide was found to be minor component of Vg and Vn from hepatopancreas and ovary extracts, respectively. We conclude that the Vn polypeptides produced by ovary are similar to those produced by hepatopancreas.     

Postadhesive Technique for Archival Paraffin Sections

We present a postadhesive protocol for adhering paraffin sections of archival material to microscope slides. Appropriately posttreated sections, subsequently processed for immunohistochemistry, remained attached to the slides and were well preserved with no signs of artifacts, such as scratching and shrinkage. The immunohistochemical staining was intense and antigen-specific without nonspecific background. Specific staining intensity was equal to that produced in untreated control sections; however, the latter became partially or fully detached from the slides. The postadhesion protocol may be used with modern techniques and is recommended for reclaiming use of otherwise unsuitable paraffin sections of archival material.     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller-Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella-free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.     

Heterologous β-phellandrene production by alginate immobilized Synechocystis sp. PCC 6803

Journal of Applied Phycology - The aim of the present work was the investigation of various cultivation conditions in order to provide a foundation for a sustainable, financially viable, and...