Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Isolation and structural determination of squalene synthase inhibitor from Prunus mume fruit

Squalene synthase plays an important role in the cholesterol biosynthetic pathway. Inhibiting this enzyme in hypercholesterolemia can lower not only plasma cholesterol but also plasma triglyceride levels. A squalene synthase inhibitor was screened from Prunus mume fruit, and then purified via sequential processes of ethanol extraction, HP-20 column chromatography, ethyl acetate extraction, silica gel column chromatography, and crystallization. The squalene synthase inhibitor was identified as chlorogenic acid with a molecular mass of 354 Da and a molecular formula of C16H18O9 based on UV spectrophotometry, 1H and 13C NMRs, and mass spectrometry. Chlorogenic acid inhibited the squalene synthase of pig liver with an IC50 level of 100 nM. Since chlorogenic acid was an effective inhibitor against the squalene synthase of an animal source, it may be a potential therapeutic agent for hypercholesterolemia.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Characterisation of the ACC oxidase activity in transgenic auxin overproducing tomato during ripening

As part of our studies on the role of auxin in regulating the ethylenebiosynthesis during fruit ripening, in this paper we describe the functionalproperties of the ACC oxidase activity extracted from transgenic tomato(Lycopersicum esculentum Mill. cv. Ailsa craig)overexpressing the tryptophan monooxygenase or iaaM protein fromAgrobacterium tumefaciens that increases the auxin levels.Maximal activity was recovered by extracting the enzyme at pH 8.0 from fruitspicked three days after the onset of the colour change. The enzyme exhibits ahalf-life of 85 min, two relative maxima at 30 and 38°C, an optimum pH of 7.9 and an apparent Km forACC of 118 M. Our results also show the first evidence of anallosteric type kinetic of the ACC oxidase activity with respect to itscosubstrate ascorbate, with an apparent Km of 12.5mM,estimated as the concentration which gave 50% Vmax.     

Lignin is linked to ethyl-carbamate formation in ume (Prunus mume) liqueur

Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. The extracts were loaded onto a C18 column, washed with water, and eluted with methanol. The (13)C-NMR spectra for the main constituents of the methanol elution fractions were consistent with those for lignin or fragments thereof. The methanol fractions were added to ume liqueur which was stored for 3 months. Relative to a control, the ethyl carbamate concentrations in the 3-month old liqueurs were found to be 1.2 and 4.6 higher for the untoasted oak-powder and the toasted oak-powder respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.     

Expression of the ACC synthase and ACC oxidase coding genes after self-pollination and incongruous pollination of tobacco pistils

In tobacco, as in other species, ethylene is produced in response to pollination. Although tobacco is a self-compatible species, it displays unilateral incongruity with other Nicotianaplants. Incongruous pollination also results in ethylene production, but this production differs depending on the pollen used and is related to the extent to which pollen tubes grow in the tobacco style. In the investigation reported here we followed the expression of the ACC synthase- and ACC oxidase-coding genes upon pollination of tobacco pistils and compared self-pollination with incongruous pollination. The pattern of expression of these genes also correlated with pollen-tube growth, although wounding alone cannot explain the results obtained. We also examined the expression of these genes upon pollination of immature tobacco pistils, in which different pollen tubes grew indistinctly inside the tobacco style and reached the ovary at the same rate. In this situation no significant differences in gene expression could be observed between the different pollinations. Ethephon, a substance that produces ethylene, could, in some cases, minimize the arrest of incongruous pollen tubes inside the style.     

G-protein-coupled alpha2A-adrenoreceptor agonists differentially alter citrus leaf and fruit abscission by affecting expression of ACC synthase and ACC oxidase

Temporal and spatial expression patterns of genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS1 and ACS2) and ACC oxidase (ACO), ACC concentration, and ethylene production in leaves and fruit of 'Valencia' orange (Citrus sinensis [L.] Osbeck) were examined in relation to differential abscission after treatment with 2-chloroethylphosphonic acid (ethephon) alone or in combination with guanfacine or clonidine, two G-protein-coupled alpha(2A)-adrenoreceptor selective agonists. Guanfacine and clonidine markedly reduced ethephon-enhanced leaf abscission, but had little effect on ethephon-enhanced fruit loosening. Ethephon-enhanced fruit and leaf ethylene production, and ACC concentration in fruit abscission zones, fruit peel, leaf abscission zones, and leaf blades were decreased by guanfacine. Guanfacine reduced ethephon-enhanced expression of ACS1 and ACO genes in leaf abscission zones and blades, but to a lesser extent in fruit abscission zones. The expression pattern of the ACS2 gene, however, was not associated with abscission. The results demonstrate that differential expression of ACS1 and ACO genes is associated with reduction of ethephon-enhanced leaf abscission by guanfacine, and suggest a link between G-protein-related signalling and abscission.     

Isolation of cDNA clones corresponding to genes differentially expressed in pericarp of mume (Prunus mume) in response to ripening, ethylene and wounding signals

Ripening of climacteric fruit is a complex developmental process that includes many changes in gene expression. Some ripening-regulated genes are responsive to ethylene and/or wounding signals. Wounding increased Pm-ACS1 expression in Prunus mume (Japanese apricot), but was negatively regulated by ethylene. However, exposure of freshly harvested mature green mume fruit to ethylene induced PmACS1 . Fifteen complementary DNA clones corresponding to messenger RNAs differentially expressed in the pericarp of P. mume fruit in response to ripening, ethylene and wounding signals were isolated by differential display. Quantitative real-time PCR analysis distinctly showed that these genes are differentially regulated. Genes that were upregulated during fruit ripening include Pm15 (cinnamyl-alcohol dehydrogenase), Pm21 (2-oxoacid-dependent dioxygenase), Pm22 (1-acyl- sn -glycerol-3-phosphate acyltransferase), Pm27 (unknown function), Pm38 (alcohol dehydrogenase), Pm41 (no homology), Pm52 (no homology), Pm65 (pectate lyase), Pm68 (expansin), Pm69 (serine carboxypeptidase) and Pm94 (alcohol acyltransferase). Expression of most of these genes was also inducible by ethylene and some of them were inducible by wounding. Pm3 (water channel protein, MIP) and Pm8 (unknown function) were downregulated during ripening. Expression of Pm71 (no homology) and Pm74 (NAC family protein) did not increase during ripening or in response to ethylene, but was upregulated in response to wounding. The possible physiological roles of these genes during ripening and in response to ethylene and wounding are discussed.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Peach (Prunus persica) fruit response to anoxia: reversible ripening delay and biochemical changes

The use of modified atmospheres has been successfully applied in different fruits to delay the ripening process and to prevent physiological disorders. In addition, during normal ripening, hypoxic areas are generated inside the fruit; moreover, anaerobic conditions may also arise during fruit post-harvest storage and handling. In consequence, the fruit is an interesting model to analyze the metabolic modifications due to changes in oxygen levels. In this work, a 72 h anoxic treatment by using an N(2) storage atmosphere was applied to peaches (Prunus persica L. Batsch) after harvest. Ripening was effectively delayed in treated fruits, preventing fruit softening, color changes and ethylene production. Metabolic changes induced by anoxia included induction of fermentative pathways, glycolysis and enzymes involved in both sucrose synthesis and degradation. Sucrose, fructose and glucose contents remained unchanged in treated fruit, probably due to sucrose cycling. Sorbitol was not consumed and citrate was increased, correlating with citric acid cycle impairment due to O(2) deprivation. Malate content was not affected, indicating compensation in the reactions producing and consuming malate. Changes in malic enzymes and pyruvate orthophosphate dikinase may provide pyruvate for fermentation or even act to regenerate NADP. After fruit transfer to aerobic conditions, no signs of post-anoxia injury were observed and metabolic changes were reversed, with the exception of acetaldehyde levels. The results obtained indicate that peach fruit is an organ with a high capacity for anoxic tolerance, which is in accord with the presence of hypoxic areas inside fruits and the fact that hypoxic pre-treatment improves tolerance to subsequent anoxia.     

The expression of alternative oxidase and uncoupling protein during fruit ripening in mango

The expression of alternative oxidase (Aox) and uncoupling proteins (Ucp) was investigated during ripening in mango (Mangifera indica) and compared with the expression of peroxisomal thiolase, a previously described ripening marker in mango. The multigene family for the Aox in mango was expressed differentially during ripening. Abundance of Aox message and protein both peaked at the ripe stage. Expression of the single gene for the Ucp peaked at the turning stage and the protein abundance peaked at the ripe stage. Proteins of the cytochrome chain peaked at the mature stage of ripening. The pattern of protein accumulation suggested that increases in cytochrome chain components played an important role in facilitating the climacteric burst of respiration and that the Aox and Ucp may play a role in post-climacteric senescent processes. Because both message and protein for the Aox and Ucp increased in a similar pattern, it suggests that their expression is not controlled in a reciprocal manner but may be active simultaneously.