The Role of ABA in the Interaction between Citrus Fruit and Penicillium digitatum.

Abscisic acid (ABA) protects citrus fruit against Penicillium digitatum infection. The global mechanisms involved in the role of ABA in the P. digitatum-citrus fruit interaction are unknown. Here, we determine the transcriptome differences between the Navelate (Citrus sinensis (L.) Osbeck) orange and its ABA-deficient mutant Pinalate, which is less resistant to infection. Low ABA levels may affect both the constitutive mechanisms that protect citrus fruit against P. digitatum and early responses to infection. The repression of terpenoid, phenylpropanoid and glutation metabolism; of oxidation-reduction processes; and of processes related to the defense response to fungus and plant hormone signal transduction may be one part of the constitutive defense reduced in the mutant against P. digitatum. Our results also provide potential targets for developing P. digitatum-citrus fruit-resistant varieties. Of those up-regulated by ABA, a thaumatin protein and a bifunctional inhibitor/LTP, which are relevant in plant immunity, were particularly remarkable. It is also worth highlighting chlorophyllase 1 (CLH1), induced by infection in Pinalate, and the OXS3 gene, which was down-regulated by ABA, because the absence of OXS3 activates ABA-responsive genes in plants.

Coordinated activation of the metabolic pathways induced by LED blue light in citrus fruit.

The biochemical changes induced by LED Blue Light (LBL) (450 nm) in Lane Late oranges were investigated. The selected quantum flux (60 µmol m-2 s-1, 2 days) was associated with resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. A holistic overview was obtained by a comparative transcriptome profile analysis, which revealed that LBL favored energy metabolism and redirected metabolic pathways toward the synthesis of diverse primary and secondary metabolism products. LBL favored reactive oxygen species homeostasis and metabolic activities involving lipid metabolism, specifically the synthesis of pigments and oxylipins, and the metabolism of carbohydrates, amino acids and indol- and alkaloid-derivatives. LBL also repressed limonene catabolism and triggered phenylpropanoid derivatives-related changes, which increased content in total flavonoids. Transferring fruit from LBL to darkness favored those processes involving amino acids, different phenylpropanoid, alkaloid and terpenoid classes, and ferrochelatase activity.

Insights into the regulation of molecular mechanisms involved in energy shortage in detached citrus fruit.

Harvested fruit undergo carbon and energy deprivation. However, the events underlying this energy-related stress in detached fruit and their involvement in cell damage have not yet been elucidated. We showed that supplementing detached sweet oranges with additional carbon or energy sources reduced peel damage, while inhibitors of energy metabolism increased it. We investigated the effect of an exogenous source of carbon (glycerol), energy (ATP), and an inhibitor of energy metabolism 2-deoxy-D-glucose (DeOGlc) + sodium iodoacetate (IAc), on the transcriptome of harvested fruit flavedo (outer peel part). ATP and Gly induced common, but also specific, alternative modes of energy metabolism by reducing the stress caused by energy shortage. They also induced shifts in energy metabolism that led to the production of the intermediates required for plant defense secondary metabolites to form. ATP and Gly triggered changes in the expression of the genes involved in cell lesion containment through a defined pathway involving hormones and redox-mediated signaling. DeOGlc + IAc had a contrasting effect on some of these mechanisms. These chemicals altered the biological processes related to membrane integrity and molecular mechanisms involving reactive oxygen species (ROS) production, and lipid and protein degradation.

Light-emitting Diode Blue Light Alters the Ability of Penicillium digitatum to Infect Citrus Fruits.

Penicillium digitatum (Pers.:Fr.) Sacc. is the main fungus causing postharvest losses in citrus fruits. Previous work showed the potential of LED blue light (LBL) in controlling P. digitatum growth. Here, we have investigated whether LBL alters the ability of this fungus to infect citrus fruits. Before fruit infection, Petri plates inoculated with the same conidia concentration were held under darkness (control) or LBL (100 µmol m-2 s-1 ) for 8 d (continuous light), or were treated with the same LBL for 3 d and then shifted to darkness for 5 d (non-continuous light). Spores from cultures exposed to continuous light showed very low capacity to germinate (1.8% respect to control) but a high viability and a similar morphology and ability to infect the fruits than spores from control cultures. The number of spores produced in plates exposed to non-continuous light was slightly lower than in control plates, but they showed much lower viability and lower capacity to infect the fruits. This effect was more likely related to aberrant morphology of spores, which formed aggregates, than to its metabolic activity or its ability to produce ethylene that might contribute to destroy natural defense barriers from the fruit.

Insights into the Molecular Events That Regulate Heat-Induced Chilling Tolerance in Citrus Fruits.

Low non-freezing temperature may cause chilling injury (CI), which is responsible for external quality deterioration in many chilling-sensitive horticultural crops. Exposure of chilling-sensitive citrus cultivars to non-lethal high-temperature conditioning may increase their chilling tolerance. Very little information is available about the molecular events involved in such tolerance. In this work, the molecular events associated with the low temperature tolerance induced by heating Fortune mandarin, which is very sensitive to chilling, for 3 days at 37°C prior to cold storage is presented. A transcriptomic analysis reveals that heat-conditioning has an important impact favoring the repression of genes in cold-stored fruit, and that long-term heat-induced chilling tolerance is an active process that requires activation of transcription factors involved in transcription initiation and of the WRKY family. The analysis also shows that chilling favors degradation processes, which affect lipids and proteins, and that the protective effect of the heat-conditioning treatment is more likely to be related to the repression of the genes involved in lipid degradation than to the modification of fatty acids unsaturation, which affects membrane permeability. Another major factor associated with the beneficial effect of the heat treatment on reducing CI is the regulation of stress-related proteins. Many of the genes that encoded such proteins are involved in secondary metabolism and in oxidative stress-related processes.

LED Blue Light-induced changes in phenolics and ethylene in citrus fruit: Implication in elicited resistance against Penicillium digitatum infection.

The objective was to investigate whether LED Blue Light (LBL) induces changes in phenolics and ethylene production of sweet oranges, and whether they participate in LBL-elicited resistance against the most important postharvest pathogen (Penicillium digitatum) of citrus fruit. The expression of relevant genes of the phenylpropanoid and ethylene biosynthetic pathways during elicitation of resistance was also determined. Different LBL (wavelength 450nm) quantum fluxes were used within the 60-630µmolm-2s-1 range. The HPLC analysis showed that the most relevant increase in phenylpropanoids occurred in scoparone, which markedly increased 3days after exposing fruits to a very high quantum flux (630µmolm-2s-1) for 18h. However, phenylpropanoids, including scoparone, were not critical factors in LBL-induced resistance. The genes involved in ethylene biosynthesis were differentially regulated by LBL. Ethylene is not involved in elicited resistance, although high LBL levels increased ethylene production in only 1h.

Influence of modified atmosphere and ethylene levels on quality attributes of fresh tomatoes (Lycopersicon esculentum Mill.).

Controlling storage atmosphere is a key factor for delaying postharvest fruit quality loss. The objective of this study is to evaluate its influence on physico-chemical, sensorial and nutritional quality attributes of two tomato fruit cultivars (Delizia and Pitenza) that respectively have a short- and long-storage life. To that end, the effect of two types of bags with different gas permeability, combined or not with an ethylene sorbent, on tomato organoleptic and nutritional properties were compared during fruit storage at 13°C. CO2 and O2 were critical factors for controlling tomato postharvest behaviour. Weight loss, firmness, color and visual quality were only affected by bag permeability just as total sugar content and acidity for Pitenza tomatoes. (trans)-2-Hexenal also appears to be related with CO2 and O2 levels. Lycopene, total phenols (TP) and ascorbic acid (AA) contents were also affected by the packaging form and the storage length. Ethylene removal in combination with MAP led to a higher content in TP and AA in the short-life tomato cultivar.

Inhibiting ethylene perception with 1-methylcyclopropene triggers molecular responses aimed to cope with cell toxicity and increased respiration in citrus fruits.

The ethylene perception inhibitor 1-methylcyclopropene (1-MCP) has been critical in understanding the hormone's mode of action. However, 1-MCP may trigger other processes that could vary the interpretation of results related until now to ethylene, which we aim to understand by using transcriptomic analysis. Transcriptomic changes in ethylene and 1-MCP-treated 'Navelate' (Citrus sinensis L. Osbeck) oranges were studied in parallel with changes in ethylene production, respiration and peel damage. The effects of compounds modifying the levels of the ethylene co-product cyanide and nitric oxide (NO) on fruit physiology were also studied. Results suggested that: 1) The ethylene treatment caused sub-lethal stress since it induced stress-related responses and reduced peel damage; 2) 1-MCP induced ethylene-dependent and ethylene-independent responsive networks; 3) 1-MCP triggered ethylene overproduction, stress-related responses and metabolic shifts aimed to cope with cell toxicity, which mostly affected to the inner part of the peel (albedo); 4) 1-MCP increased respiration and drove metabolism reconfiguration for favoring energy conservation but up-regulated genes related to lipid and protein degradation and triggered the over-expression of genes associated with the plasma membrane cellular component; 5) Xenobiotics and/or reactive oxygen species (ROS) might act as signals for defense responses in the ethylene-treated fruit, while their uncontrolled generation would induce processes mimicking cell death and damage in 1-MCP-treated fruit; 6) ROS, the ethylene co-product cyanide and NO may converge in the toxic effects of 1-MCP.

Effect of LED Blue Light on Penicillium digitatum and Penicillium italicum Strains.

Studies on the antimicrobial properties of light have considerably increased due in part to the development of resistance to actual control methods. This study investigates the potential of light-emitting diodes (LED) blue light for controlling Penicillium digitatum and Penicillium italicum. These fungi are the most devastating postharvest pathogens of citrus fruit and cause important losses due to contaminations and the development of resistant strains against fungicides. The effect of different periods and quantum fluxes, delaying light application on the growth and morphology of P. digitatum strains resistant and sensitive to fungicides, and P. italicum cultured at 20°C was examined. Results showed that blue light controls the growth of all strains and that its efficacy increases with the quantum flux. Spore germination was always avoided by exposing the cultures to high quantum flux (700 µmol m(-2) s(-1) ) for 18 h. Continuous light had an important impact on the fungus morphology and a fungicidal effect when applied at a lower quantum flux (120 µmol m(-2) s(-1) ) to a growing fungus. Sensitivity to light increased with mycelium age. Results show that blue light may be a tool for P. digitatum and P. italicum infection prevention during handling of citrus fruits.

Cell wall modifications and ethylene-induced tolerance to non-chilling peel pitting in citrus fruit.

Non-chilling peel pitting (NCPP), a storage disorder resulting in the formation of depressed areas in the peel of many citrus cultivars, is reduced by ethylene treatments. We hypothesized that this effect may be associated with biochemical changes of cell wall components. Therefore, we extracted cell wall material from albedo and flavedo tissues of 'Navelate' oranges stored in air, conditioned with ethylene (2µLL(-1)) for 4 days and subsequently transferred to air, or continuously stored in an ethylene-enriched atmosphere (2µLL(-1)). Uronic acids and neutral sugars were extracted into five fractions enriched in specific wall polymers namely water-, CDTA-, Na2CO3-, and 1 and 4M KOH-soluble fractions. Pectin insolubilization was found in control fruit at long storage times. Ethylene treatments, alleviating NCPP, increased polyuronide solubility in the albedo and had a slight effect on the flavedo. Ethylene-treated fruit showed greater content of water-soluble neutral sugars and a larger proportion of hemicelluloses readily extractable with 1M KOH, with a concomitant reduction in the 4M KOH-soluble fraction. This suggests that the protective role of ethylene on NCPP is associated with an increased solubilization of the wall of albedo cells.

The Citrus ABA signalosome: identification and transcriptional regulation during sweet orange fruit ripening and leaf dehydration.

The abscisic acid (ABA) signalling core in plants include the cytosolic ABA receptors (PYR/PYL/RCARs), the clade-A type 2C protein phosphatases (PP2CAs), and the subclass III SNF1-related protein kinases 2 (SnRK2s). The aim of this work was to identify these ABA perception system components in sweet orange and to determine the influence of endogenous ABA on their transcriptional regulation during fruit development and ripening, taking advantage of the comparative analysis between a wild-type and a fruit-specific ABA-deficient mutant. Transcriptional changes in the ABA signalosome during leaf dehydration were also studied. Six PYR/PYL/RCAR, five PP2CA, and two subclass III SnRK2 genes, homologous to those of Arabidopsis, were identified in the Citrus genome. The high degree of homology and conserved motifs for protein folding and for functional activity suggested that these Citrus proteins are bona fide core elements of ABA perception in orange. Opposite expression patterns of CsPYL4 and CsPYL5 and ABA accumulation were found during ripening, although there were few differences between varieties. In contrast, changes in expression of CsPP2CA genes during ripening paralleled those of ABA content and agreeed with the relevant differences between wild-type and mutant fruit transcript accumulation. CsSnRK2 gene expression continuously decreased with ripening and no remarkable differences were found between cultivars. Overall, dehydration had a minor effect on CsPYR/PYL/RCAR and CsSnRK2 expression in vegetative tissue, whereas CsABI1, CsAHG1, and CsAHG3 were highly induced by water stress. The global results suggest that responsiveness to ABA changes during citrus fruit ripening, and leaf dehydration was higher in the CsPP2CA gene negative regulators than in the other ABA signalosome components.

Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant.

Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and -independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage.

Ultrastructural and histochemical analysis reveals ethylene-induced responses underlying reduced peel collapse in detached citrus fruit.

Fruits from many citrus cultivars develop depressed areas in the flavedo (outer part of the peel) and albedo (inner part) following detachment. Although ultrastructural analysis may provide important information about multiple plant responses to stresses and external stimuli at the cell and tissue levels, and despite the proved efficacy of ethylene in reducing peel damage in citrus fruit, cytological responses of this horticultural crop to protective ethylene concentrations have not yet been reported. We show that applying high ethylene levels (2 µL L(-1) for 14 days) causes sublethal stress as it favored the alteration of cuticle, vacuole, middle lamella and primary wall, especially in the albedo cells, but reduced peel collapse in detached mature "Navelate" oranges (C. sinensis, L. Osbeck) held under nonstressful environmental conditions (22°C and 90-95% RH). Ethylene did not induce relevant changes in lignification but favored the deposition of pectic exudates and the release of sugars from degradation of cell polysaccharides including starch, cellulose, and pectins. In contrast, inhibiting ethylene perception by applying 1-methylcyclopropene (1-MCP) reduced these ethylene-related responses and favored degradation of cell membranes and peel damage. The overall results reflect that mature oranges tolerate high ethylene levels that might favor the activation of defense responses involving oxidative-stress related mechanisms and recycling of nutrients and carbon supply to enable cells to sustain respiration and cope with carbon deprivation stress caused by detachment.

Influence of postharvest treatments on quality, carotenoids, and abscisic acid content of stored "spring belle" peach (prunus persica) fruit.

The influence of four postharvest treatments, 1-methylcyclopropene (1-MCP), carbon dioxide (CO2), and nitrogen (N2), followed by fruit storage at 10 degrees C or of hydrocooling (H2O) at 1 degrees C, followed by storage at 0 degrees C on fruit quality, carotenoids, and abscisic acid (ABA) content as well as on ethylene and carbon dioxide production of "Spring Belle" peach fruits, has been examined. Ethylene production was reduced by all the treatments and raised after transfer the fruits at 20 degrees C, their ethylene production in general being lower than that of fruits continuously held at 20 degrees C. Nevertheless, 1-MCP removal enhanced the rise in ethylene occurring at 20 degrees C by the end of storage. Those changes were likely related to fruit softening but not to changes in color or in the soluble solid content (SSC). HPLC analyses showed a relative high content of xanthophylls, particularly violaxanthin. In fruits maintained in air at 20 degrees C, violaxanthin and beta-carotene contents decreased while beta-criptoxanthin increased. ABA content showed a great increase in 1-MCP and significant decrease in carbon dioxide and hydrocooling treated peaches. The results indicated hydrocooling, in combination with low temperature storage, as the best treatment maintaining fruit firmness due to the lowered respiration rate and the content of relevant carotenoids.

Carbohydrate metabolism as related to high-temperature conditioning and peel disorders occurring during storage of citrus fruit.

The aim of this research was to understand the involvement of the carbohydrate metabolism in physiological disorders occurring during the postharvest storage of citrus fruit. These disorders, manifested in the rind, depreciate fruit quality and often originate important losses. There has been increasing interest in the use of nonharmful treatments, such as high-temperature conditioning, to avoid citrus peel damage during fruit storage at low temperature in chilling-sensitive cultivars, but their influence in postharvest disorders occurring at nonchilling temperatures and the mechanisms related to them are poorly understood. The data obtained showed that heat conditioning (3 days/37 degrees C) increases the chilling tolerance of cv. Navelate fruit and favored sucrose, but not hexoses, accumulation and its maintenance after the fruit was transferred to low temperature. This effect was related to heat-induced increase in the activities of the sucrose-synthesizing enzymes sucrose phosphate synthase (SPS) and sucrose synthase (SS). Furthermore, sucrose levels and the activities of both enzymes were higher in cv. Pinalate oranges, a chilling-tolerant spontaneous abscisic acid deficient mutant of Navelate. In contrast, carbohydrates appeared not to be involved in the susceptibility of oranges to rind staining, a physiological disorder different from chilling injury, which mainly occurred at a nonchilling temperature (12 degrees C) and was not reduced by heat conditioning. The effect of low temperature in SS and SPS activities was less than that of high temperature, which might be related to the lower changes occurring in sucrose during fruit storage at 2 degrees C.

Active oxygen detoxifying enzymes and phenylalanine ammonia-lyase in the ethylene-induced chilling tolerance in citrus fruit.

The effects of applying ethylene (2 microL x L(-)(1)) during cold storage of Fortune mandarins on the development of chilling-induced peel damage and on changes in the activities of the enzymes of the antioxidant system, superoxide dismutase, catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, and on phenylalanine ammonia-lyase (PAL) have been investigated. Chilling damage was reduced by applying ethylene during fruit storage at 1.5 degrees C. PAL activity increased in response to cold stress and was higher in fruit held under ethylene than under air during the whole storage period, whereas CAT was temporarily higher in ethylene-treated fruit. In contrast, the activities of the other enzymes were not increased by ethylene. The global results suggest that the ethylene-induced chilling tolerance in Fortune mandarins might be due to increased PAL and CAT activities.