Retrotransposon promoter of Ruby1 controls both light- and cold-induced accumulation of anthocyanins in blood orange.

Blood orange is generally recognized to accumulate anthocyanins in its fruit pulp in a cold-inducible manner. We observed that the fruit peel of blood orange can also accumulate anthocyanins under ample light conditions. Interestingly, purple pummelo can accumulate anthocyanins only in its fruit peel but not in its pulp. The mechanism underlying the tissue specificity of anthocyanin accumulation in citrus is unknown. Here, we show that the active promoter of Ruby1, a key activator of anthocyanin biosynthesis, is also light inducible in addition to its already known cold inducibility in blood orange. Electrophoretic mobility shift assays and transient expression assays showed that HY5 positively regulated the transcription of Ruby1 by binding to the G-box motif (CACGTC). The tissue specificity of anthocyanin accumulation in the peel of purple pummelo may be due to the lack of a low temperature responsive element and a MYC binding site, which were shown to be involved in cold inducibility of CsRuby1 in blood orange by insertion of a long terminal repeat type retrotransposon in the promoter. These results bring new insights into the regulatory mechanism of anthocyanin biosynthesis in response to environmental stimuli and provide cis-elements for genetic improvement of anthocyanin-stable fruits rich in antioxidant metabolites.

Effect of Light-Emitting Diodes and Ultraviolet Irradiation on the Soluble Sugar, Organic Acid, and Carotenoid Content of Postharvest Sweet Oranges (Citrus sinensis (L.) Osbeck).

Mature 'Hamlin' sweet oranges (Citrus sinensis (L.) Osbeck) were irradiated using light-emitting diodes (LEDs) and ultraviolet (UV) light for six days after harvest. Based on evaluation of the basic ripening parameters of fruits, the contents of soluble sugars, organic acids, and carotenoids were analyzed (in pulps) on the sixth day by high-performance liquid chromatography (HPLC). The results showed that LED and UV irradiation not only accelerated orange ripening but also caused significant changes in the soluble sugar, organic acid, and carotenoid content. Compared with fruit subjected to dark shade (DS) treatment, the total soluble sugar, fructose, and glucose contents increased significantly in UV-treated (UVA, UVB, and UVC) fruits, while the sucrose content increased remarkably in white light, UVB, and UVC-treated fruits (p < 0.05). UV treatment was associated with inducing the largest effect on the total soluble sugar content. Except for UVB, other types of light notably induced an accumulation of the total organic acid content, none but blue light and red light markedly induced citric acid accumulation (p < 0.05). Interestingly, only the red light and dark shade treatments had markedly positive effects in terms of inducing carotenoid accumulation, including the total carotenoid, isolutein, zeaxanthin, lutein, neoxanthin, all-trans-violaxanthin, phytofluene, cis-ζ-carotene, and ß-carotene concentrations. Other light treatments had significantly negative effects on carotenoid accumulation (p < 0.05). Therefore, soluble sugar, organic acid, and carotenoid accumulation in sweet oranges vary depending on the levels of UV and LED irradiation. Appropriate light irradiation is a potentially effective way to maintain or improve postharvest fruit quality.

Metabolism in orange fruits is driven by photooxidative stress in the leaves.

In plants, stress signals propagate to trigger distant responses and thus stress acclimation in non-exposed organs. We tested here the hypothesis that leaves submitted to photooxidative stress may influence the metabolism of nearby fruits and thus quality criteria. Leaves of orange trees (Citrus sinensis (L.) Osbeck cv. 'Navelate') were acclimated to shade for 1 week and then submitted to full (FL) and medium light (ML) conditions. As expected, photoinhibition was detected in leaves of both FL and ML treatments as revealed by stress indicators (Fv /Fm , Performance Index) for at least 99 h after treatments. In the fruits near the stressed leaves, we then determined the activities of enzymes related to oxidative stress, superoxide dismutase, catalase and the enzymes of the ascorbate (AA)/glutathione cycle, as well as the contents in sugars, organic acids and carotenoids. Ascorbate peroxidase and monodehydroascorbate reductase activities in the pulp of fruits were dramatically higher in both treatments when compared to the control. AA and total sugars were not affected by the photooxidative stress. However, the FL treatment resulted in a 16% increase in total organic acids, with succinic acid being the major contributor, a shift towards less glucose + fructose and more sucrose, and a 15% increase in total carotenoids, with cis-violaxanthin being the major contributor. Our observations strongly suggest the existence of a signal generated in leaves in consequence of photooxidative stress, transmitted to nearby fruits. Exploiting such a signal by agronomic means promises exciting perspectives in managing quality criteria in fruits accumulating carotenoids.

Synergistic effects of ultrasound and ß-d-glucosidase in aroma of orange juice.

The synergistic effects of ultrasound and ß-d-glucosidase in aroma of orange juice were investigated. ß-d-Glucosidase significantly increased the content of ester, aldehyde, alcohol, terpene, acid, and phenol, and insignificantly increased the ketone content in orange juice. Enzyme-treated orange juice, compared with fresh untreated orange juice, was found to contain 15 novel aroma compounds, whereas three aroma compounds disappeared. Ultrasound improved the enzymatic action and the retention of more active flavors in juice than treatment with enzyme alone. However, simultaneous ultrasound and enzyme treatment decreased aroma quality. Therefore, the choice of the mode of ultrasound treatment is highly important. The present investigation will provide a reference for aroma-enhancing application of ultrasound combined with ß-D-glucosidase. PRACTICAL APPLICATION: The study supplies a reference method for the aromatization of fruit juice.

Blue LED light induces regreening in the flavedo of Valencia orange in vitro.

In the present study, the effects of blue LED light on the regreening of citrus fruit were investigated in an in vitro system of Valencia orange flavedos. The results showed that blue LED light irradiation induced regreening in the flavedos. After four-week culture in vitro, the flavedos exhibited obviously green color in the blue LED light treatment, while the flavedos in the control were still in orange color. During the regreening process, the blue LED light treatment induced chlorophyll accumulation, and substantially altered the carotenoid composition in the flavedos. Compared with the control, the content of 9-cis-violaxanthin was decreased, while the contents of lutein, ß-carotene, and all-trans-violaxanthin were increased by blue LED light. In addition, gene expression results showed that the up-regulation of CitLCYe and down-regulation of CitLCYb2 by blue LED light led to a shift from ß,ß-branch to ß,ε-branch of the carotenoid biosynthetic pathway.

Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis.

The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

Intensification of polyphenols extraction from orange peels using infrared as a novel and energy saving pretreatment.

This study aims to evaluate the impact of a nonconventional pretreatment technique "infrareds free solvent" on the intensification of polyphenols extraction from orange peels. Orange peels were pretreated with infrared heating using a ceramic infrared transmitter from 5 to 25 min at 50 °C. After the addition of the solvent on the pretreated peels, ultrasound treatment was applied on the mixture using an ultrasound generator connected to a titanium ultrasound probe, from 5 to 30 min, at 50 °C. Results showed that the application of ultrasounds on untreated peels enhanced the extraction of polyphenols by 62.5% compared to the conventional solid-liquid extraction. Twenty minutes of infrared pretreatment improved the extraction of polyphenols by 47% with solid-liquid extraction, and 112% with ultrasounds after 30 min compared to solid-liquid extraction from untreated peels. Different combinations of infrared pretreatment and ultrasound assisted extraction were then applied on orange peels. The most advantageous combination in terms of energy consumption and polyphenols extraction has been found for a 20 min infrared pretreatment time and 5 min ultrasound assisted extraction of polyphenols. PRACTICAL APPLICATION: Orange peels are valuable sources of natural antioxidants such as polyphenols. Ultrasound-assisted extraction can improve the extraction of polyphenols compared to conventional solid-liquid extraction. To intensify the extraction process, infrared heating can be used as a simple, low cost, and energy saving method. The combined effect of "infrareds free solvent" and ultrasounds allowed the extraction of the highest yields of polyphenols with a high antiradical capacity and a low energy consumption in comparison to conventional extraction.

Effects of electron-beam irradiation on the quality characteristics of mandarin oranges (Citrus unshiu (Swingle) Marcov) during storage.

Consumption and global utilization of mandarins is on the rise due to its easy-to-peel attribute compared to other citrus fruits. Mandarins are irradiated as an effective phyto-sanitary treatment for trade requirements. In this study, mandarins were irradiated with 0.4 and 1 kGy electron-beam as approved by United States Department of Agriculture (USDA), and changes in microbial and physicochemical characteristics during storage were monitored at 4 °C for 15 d. Microbial proliferation during storage was noticeable in non-irradiated fruits, whereas irradiated mandarins showed dose-dependent inhibition up to 15 d. Irradiation did not affect its moisture, total phenolics, and vitamin C. However, hardness, organic acids, hesperidin, radical scavenging activity, reducing and free sugars significantly decreased in all samples after 5 d of storage. Although, irradiation at 0.4 and 1 kGy could not prevent changes in stored mandarins, 0.4 kGy did not affect the major constituents and physical quality of mandarins, while providing microbial decontamination.

CsPLDalpha1 and CsPLDgamma1 are differentially induced during leaf and fruit abscission and diurnally regulated in Citrus sinensis.

Understanding leaf and fruit abscission is essential in order to develop strategies for controlling the process in fruit crops. Mechanisms involved in signalling leaf and fruit abscission upon induction by abscission agents were investigated in Citrus sinensis cv. 'Valencia'. Previous studies have suggested a role for phospholipid signalling; hence, two phospholipase D cDNA sequences, CsPLDalpha1 and CsPLDgamma1, were isolated and their role was examined. CsPLDalpha1 expression was reduced in leaves but unaltered in fruit peel tissue treated with an ethylene-releasing compound (ethephon), or a fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP). By contrast, CsPLDgamma1 expression was up-regulated within 6 h (leaves) and 24 h (fruit peel) after treatment with ethephon or CMNP, respectively. CsPLDalpha1 expression was diurnally regulated in leaf blade but not fruit peel. CsPLDgamma1 exhibited strong diurnal oscillation in expression in leaves and fruit peel with peak expression around midday. While diurnal fluctuation in CsPLDalpha1 expression appeared to be light-entrained in leaves, CsPLDgamma1 expression was regulated by light and the circadian clock. The diurnal expression of both genes was modulated by ethylene-signalling. The ethephon-induced leaf abscission and the ethephon- and CMNP-induced decrease in fruit detachment force were enhanced by application during rising diurnal expression of CsPLDgamma1. The results indicate differential regulation of CsPLDalpha1 and CsPLDgamma1 in leaves and fruit, and suggest possible roles for PLD-dependent signalling in regulating abscission responses in citrus.

Differentiation Using Microwave Plasma Torch Desorption Mass Spectrometry of Navel Oranges Cultivated in Neighboring Habitats.

The molecular fingerprinting of intact fruit samples combined with statistical data analysis can allow the assessment of fruit quality and location of origin. Herein, microwave plasma torch desorption ionization mass spectrometry (MPT-MS) was applied to produce molecular fingerprints for the juice sac and exocarp of navel oranges cultivated in three closely located habitats, and the mass spectrometric fingerprints were differentiated by principal component analysis (PCA). Because of the relatively high temperature and high ionization efficiency of MPT, the volatile aroma compounds and semivolatile chemicals in the navel oranges were sensitively detected and confidently identified by collision induced dissociation (CID). The limit of detection (LOD) of MPT-MS for vanillin was 0.119 µg/L, with the relative standard deviation (RSD, n = 10) of 1.7%. The results showed that MPT-MS could be a powerful analytical platform for the sensitive molecular analysis of fruits at molecular level with high chemical specificity, allowing differentiation between the same sorts grown in neighboring habitats.

Preparation, characterization and evaluation of adsorptive properties of orange peel based activated carbon via microwave induced K2CO3 activation.

This work explores the feasibility of orange peel, a citrus processing biomass as an alternative precursor for preparation of activated carbon (OPAC) via microwave assisted K(2)CO(3) activation. The operational parameters, chemical impregnation ratio, microwave power and irradiation time on the carbon yield and adsorption capability were investigated. The virgin characteristics of OPAC were examined by pore structural analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, nitrogen adsorption isotherm, elemental analysis, surface acidity/basicity and zeta potential measurement. The optimum conditions resulted in OPAC with a monolayer adsorption capacity of 382.75 mg/g for methylene blue and carbon yield of 80.99%. The BET surface area, Langmuir surface area and total pore volume were identified to be 1104.45 m(2)/g, 1661.04 m(2)/g and 0.615 m(3)/g, respectively. Equilibrium data were simulated using the Langmuir, Freundlich, Dubinin-Radushkevich, Redlich-Peterson, and Toth isotherms, and kinetic data were fitted to the pseudo-first-order, pseudo-second-order and Elovich kinetic models.

Effect on orange juice of batch pasteurization in an improved pilot-scale microwave oven.

The effects on orange juice batch pasteurization in an improved pilot-scale microwave (MW) oven was evaluated by monitoring pectin methyl-esterase (PME) activity, color, carotenoid compounds and vitamin C content. Trials were performed on stirred orange juice heated at different temperatures (60, 70, 75, and 85 degrees C) during batch process. MW pilot plant allowed real-time temperature control of samples using proportional integrative derivative (PID) techniques based on the infrared thermography temperature read-out. The inactivation of heat sensitive fraction of PME, that verifies orange juice pasteurization, showed a z-value of 22.1 degrees C. Carotenoid content, responsible for sensorial and nutritional quality in fresh juices, decreased by about 13% after MW pasteurization at 70 degrees C for 1 min. Total of 7 carotenoid compounds were quantified during MW heating: zeaxanthin and beta-carotene content decreased by about 26%, while no differences (P < 0.05) were found for beta-cryptoxanthin in the same trial. A slight decrease in vitamin C content was monitored after MW heating. Results showed that MW heating with a fine temperature control could result in promising stabilization treatments.

Ammonium transport and CitAMT1 expression are regulated by light and sucrose in Citrus plants.

Here the isolation and characterization of CitAMT1 cDNA from citrange Troyer (Citrus sinensis L. OsbeckxPoncirus trifoliata Blanco) is reported, suggesting that this belongs to the AMT gene family, which is involved in the high-affinity transport system (HATS). Results show that in Citrus plants, the HATS is much more dependent on the light conditions and C status of the roots than the low-affinity transport system. Most importantly, a strong correlation was found between the regulation of both HATS activity and CitAMT1 expression. CitAMT1 expression is sucrose-stimulated and may account for the regulation of NH(4)(+) HATS. Furthermore, a similar link was also recorded with photosynthetic activity in the shoots, suggesting that the variations in production and transport of photosynthates to the roots are responsible for the diurnal changes of both CitAMT1 expression and NH(4)(+) HATS activity. On the other hand, results indicate that the effect of stimulating light on CitAMT1 expression and NH(4)(+) HATS activity is independent of the circadian rhythm. Finally, CitAMT1 expression seems to be specifically stimulated by sucrose, suggesting that sucrose is a pivotal signal governing both assimilate partitioning from source organs and assimilate utilization in sink organs.