Physiology of pepper fruit and the metabolism of antioxidants: chloroplasts,mitochondria and peroxisomes

Background and Aims Pepper (Capsicum annuum) contains high levels of antioxidants, such as vitamins A and C and flavonoids. However, information on the role of these beneficial compounds in the physiology of pepper fruit remains scarce. Recent studies have shown that antioxidants in ripe pepper fruit play a key role in responses to temperature changes, and the redox state at the time of harvest affects the nutritional value for human consumption. In this paper, the role of antioxidant metabolism of pepper fruit during ripening and in the response to low temperature is addressed, paying particular attention to ascorbate, NADPH and the superoxide dismutase enzymatic system. The participation of chloroplasts, mitochondria and peroxisomes in the ripening process is also investigated.Scope and Results Important changes occur at a subcellular level during ripening of pepper fruit. Chloroplasts turn into chromoplasts, with drastic conversion of their metabolism, and the role of the ascorbate–glutathione cycle is essential. In mitochondria from red fruits, higher ascorbate peroxidase (APX) and Mn-SOD activities are involved in avoiding the accumulation of reactive oxygen species in these organelles during ripening. Peroxisomes, whose antioxidant capacity at fruit ripening is substantially affected, display an atypical metabolic pattern during this physiological stage. In spite of these differences observed in the antioxidative metabolism of mitochondria and peroxisomes, proteomic analysis of these organelles, carried out by 2-D electrophoresis and MALDI-TOF/TOF and provided here for the first time, reveals no changes between the antioxidant metabolism from immature (green) and ripe (red) fruits.Conclusions Taken together, the results show that investigation of molecular and enzymatic antioxidants from cell compartments, especially chloroplasts, mitochondria and peroxisomes, is a useful tool to study the physiology of pepper fruit, particularly in the context of expanding their shelf-life after harvest and in maintaining their nutritional value.     

Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

Using a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3?L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17?g/L), corn steep liquor (69.25?g/L), and casamino acids (80.68?g/L) in a stirred tank bioreactor under operating conditions constant at 635?rpm, a temperature of 26°C, and pH 3.3 produced 1.25?×?10⁸?blastospores (bls)/ml, with 93% viability after 120?h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.     

Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure-function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the ¹⁴C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer.     

Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.     

Seed to seedling transitions in successional habitats across a tropical landscape

Recognition that tree recruitment depends on the balance between seed arrival and seedling survival has led to a surge of interest in seed‐dispersal limitation and seedling‐establishment limitation in primary forests. Virtually unaddressed are comparisons of this balance in mature and early successional habitats. We assessed seed rain and seedling recruitment dynamics of tree species in primary forest, secondary forest and pasture released from grazing in a tropical agricultural landscape. Seed to seedling ratios (seed effectiveness; Φ_i) for 43 species in southern Mexico determined differences in the extent to which seeds produced seedlings by habitat, life history, and dispersal mode. Reproductive potential as estimated by the transition from seed rain to seedling recruitment, differed by habitats, and varied dramatically by life history and dispersal mode. Expected recruit densities (E_it) were higher for animal‐dispersed than wind‐dispersed species, and for non‐pioneer than pioneer species. Non‐pioneers and animal‐dispersed species had higher expected relative recruit abundance (ε_it) in primary forest (median of 4 seeds recruit^?1) whereas in secondary forest wind‐dispersed pioneers had the highest expected relative recruit abundance (median of 16 seeds per recruit). In pastures, wind‐dispersed pioneer species were most successful with many more seeds per recruit (median of 291) than both forest habitats. Seeds per recruit (Φ_i) appeared to decrease with increase in seed mass for 43 species for which data were available (r = –0.55, P < 0.001). This was associated with a negative correlation of Φ_i with seed size in primary forest (r = –0.50, P = 0.08 for 13 species); Φ_i was not correlated with seed size in secondary forest (n = 16) or pasture (n = 14). Metrics of seeds per recruit, expected recruit density and expected relative recruit abundance dramatically illustrate differences in barriers to recruitment in successional habitats.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves. An erratum to this article can be found at     

Atorvastatin modifies the protein profile of circulating human monocytes after an acute coronary syndrome

Aggressive treatment with high‐dose atorvastatin reduces more effectively the incidence of cardiovascular events than moderate statin therapy. The mechanism of this benefit has not been fully elucidated. In order to know the potential effects of statin treatment on the protein expression of circulating monocytes in acute coronary syndrome (ACS) patients, a proteomic analysis of these cells was carried out by 2‐DE and MS. Twenty‐five patients with non‐ST‐elevation acute coronary syndrome (NSTEACS) were randomized, the fourth day after admission, to receive ATV 80 mg/dL (n = 14) or conventional treatment (CT) (n = 11), for two months. Blood was withdrawn at the end of the treatment, and monocytes were extracted for proteomic analysis and their protein expression patterns determined. Age, sex, total cholesterol, LDL, HDL, triglycerides, body mass index, presence of hypertension, diabetes, and smoking status were not significantly different between the two groups of patients. The expression of 20 proteins was modified by intensive ATV. Among the most relevant results stand out the normalization by intensive ATV treatment of the expression of proteins that modulate inflammation and thrombosis such as protein disulfide isomerase ER60 (PDI), Annexin I, and prohibitin, or that have other protective effects as HSP‐70. Thus, this approach shed light at the molecular level of the beneficial mechanisms of anti‐atherothrombotic drugs.