Temperature and ultra low oxygen effects and involvement of ethylene in chilling injury of 'Rojo Brillante' persimmon fruit.

The effects of storage temperature, inhibition of ethylene action by treatment with 1-methylcyclopropene (1-MCP) and ultra low oxygen (ULO) atmosphere on chilling injury (CI), fruit firmness and ethylene production in the astringent 'Rojo Brillante' persimmon fruit were investigated. CI symptoms were manifested as a very dramatic loss of firmness after fruit transfer from cold storage to shelf-life conditions (18 °C). During cold storage, fruit softening appeared more rapidly in fruit stored at the intermediate temperature of 10 °C than at 1 °C or 14.5 °C. Ethylene production increased with storage time at the chilling temperature (1 °C) but a sharp increase took place upon fruit transfer from 1 °C to ambient temperature. This ethylene increase was accompanied by a loss of fruit firmness associated with chilling damage development. A pre-treatment with the competitive inhibitor of ethylene action 1-MCP, at 1 µL/L, reduced firmness loss and mitigated CI damage but considerably increased ethylene production in fruit transferred to shelf-life conditions after a prolonged cold storage period. Collectively, these results suggest a role of ethylene in the reduction of flesh firmness and consequently in the induction of CI in persimmon fruit. Moreover, ethylene exerts a negative feedback regulation of cold-induced ethylene biosynthesis. Storage of 'Rojo Brillante' persimmon fruit under ULO (1.3-1.8% O2, v/v) atmosphere did not affect the incidence of CI but reduced fruit astringency, suggesting that ULO may be an alternative postharvest storage system for 'Rojo Brillante' persimmon fruit.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Phenylalanine ammonia-lyase as related to ethylene in the development of chilling symptoms during cold storage of citrus fruits.

Low-temperature, nonfreezing, storage induces pitting and necrosis in the flavedo tissue of chilling susceptible citrus fruits. In this study the role of ethylene and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in the cold-induced citrus peel damage has been investigated. It has been shown that increasing PAL activity by applying ethylene at a nonchilling temperature did not cause fruit damage or reduce the incidence of this peel disorder when fruits were subsequently held at a chilling temperature (2 degrees C). The cold-induced peel damage was enhanced by applying inhibitors of PAL activity and ethylene synthesis and action. These results indicate that the induction of PAL and ethylene during fruit cold storage, but not before, plays a role in reducing the development of chilling symptoms. The cold-induced PAL activity was reduced by inhibitors of ethylene production, but inhibitors of ethylene action exerted little effect on the activation of this enzyme. Therefore, the activation of PAL may be dependent on ethylene but also an independent cold signal apparently related to the cold-induced peel damage.

Accumulation of PAL transcript and PAL activity as affected by heat-conditioning and low-temperature storage and its relation to chilling sensitivity in mandarin fruits.

The effects of different periods of heating at 37 degrees C on phenylalanine ammonia-lyase (PAL) and how this relates to chilling tolerance was investigated in fruits of the chilling-sensitive Fortune mandarin. All effective heat-conditioning treatments caused an early and transient increase in PAL mRNA and PAL activity. Conditioning fruits at 37 degrees C for 1 or 2 days prevented the manifestation of chilling symptoms but not the accumulation of PAL mRNA and PAL activity observed in untreated fruits. In fruits conditioned for 3 days, cold-induced damage and PAL activity were also suppressed but not the accumulation of PAL transcript upon subsequent storage at 2 degrees C. Storage of 3-day-heated fruits at a nonchilling temperature (12 degrees C) induced an early and transient increase in both PAL mRNA and PAL activity. High levels of PAL transcript and PAL activity were detected in freshly harvested fruits of a chilling-resistant mandarin (Hernandina) that decreased upon cold storage at 2 degrees C in heat-treated and nontreated fruits. These results indicate that sensitivity of mandarins to chilling correlates with low constitutive levels of PAL mRNA and PAL activity and with the inducibility of both upon exposure to low temperatures.

Carbohydrate content and metabolism as related to maturity and chilling sensitivity of cv. Fortune mandarins.

Fruits of cv. Fortune mandarin were periodically harvested throughout the ripening period to evaluate changes in carbohydrate content and metabolism in flavedo tissue and to determine the potential role of carbohydrates in the tolerance of citrus fruit to chilling injury (CI). Sucrose showed little change in the flavedo during the season, but fructose and glucose increased, in nearly equal amounts, throughout the fall and winter, reaching a maximum in January. Starch levels were less abundant than soluble carbohydrates and rose continuously until March. Sucrose phosphate synthase (SPS; EC 4.1.14) activity decreased from December throughout ripening. Changes in sucrose synthase (SS; EC 2.4.1.13) and acid and alkaline invertase (Inv; EC 3.2.1.26) activities correlated with changes in the reducing sugars, but acid invertase was less active than the other sucrose-metabolizing enzymes. Carbohydrate changes in the flavedo of Fortune mandarins with fruit maturity appear not to be related to the chilling tolerance of fruits during cold storage.

Catalase in the heat-induced chilling tolerance of cold-stored hybrid Fortune mandarin fruits.

Hybrid Fortune mandarins developed chilling injury (CI) upon cold storage, unless the fruits were conditioned at 37 degrees C for 3 days before they were held at low temperature. This heat treatment induced 2.5-, 1.2-, and 1.4-fold increases in the activities of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD), respectively, and reduced the activity of glutathione reductase (GR). The differences in the activities afforded by the heat treatment were, in general, maintained during cold storage. However, SOD levels in nonconditioned Fortune fruits exhibiting CI were similar to those of conditioned fruits stored for 0 or 6 weeks at 2 degrees C. No difference between APX activity in the conditioned and nonconditioned fruits stored for 6 weeks at 2 degrees C was found. The data indicate that CAT may be a major antioxidant enzyme operating in the heat-induced chilling tolerance of cold-stored Fortune mandarin fruits.

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins.

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37 degrees C for 6 hours followed by 4 days at 2.5 or 12.5 degrees C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5 degrees C, but not to 12.5 degrees C, a nonchilling temperature. Exposure to 37 degrees C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5 degrees C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37 degrees C and held at the nonchilling temperature of 12.5 degrees C. There was no relationship between abscisic acid content following exposure to 12.5 or 37 degrees C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42 degrees C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42 degrees C.

GLC analysis of thiabendazole residues in citrus fruit.

Analysis of thiabendazole (TBZ) residues in citrus fruit is performed using a bonded phase, SE-54, fused-silica capillary column. The fungicide is extracted with hexane: ethyl acetate (90:10, v/v) at high pH and, after a short cleanup, determined by gas-liquid chromatography with a nitrogen-phosphorus detector (NPD). Recoveries through the method are always higher than 80% and the limit of detection is 0.01 mg/Kg. TBZ residues are determined in whole fruit, peel, and pulp of "Washington Navel" oranges and "Hernandina" clementines treated with 1500 mg/L fungicide. Residues found in these samples and their changes during storage are reported. TBZ analysis in samples with high residue levels (peel and whole fruit) is also carried out by direct determination in the crude extracts. Results obtained with this shorter method are similar to those of the former proposed method.

Gas chromatographic determination of fenpropimorph residues in citrus fruit.

A rapid gas chromatographic method for determining fenpropimorph residues in citrus fruit is reported. The fungicide is extracted with hexane after pH adjustment of the fruit homogenate. A short liquid-liquid partitioning process is performed before gas chromatography on an OV-17 column with nitrogen-phosphorus specific detection. The limit of detection of the method was 0.01 mg/kg, based on a 25 g sample. Recovery was always higher than 70%. Fenpropimorph residues in "Washington Navel" oranges and "Hernandina" clementine fruits dipped in a 1500 mg/L fungicide solution were determined. The fungicide remains mainly in the peel, with levels less than 0.1 mg/kg in the pulp. Fungicide residues in the peel decrease during storage, mainly in Washington Navel peel, where values decreased from 5.2 to 2.8 mg/kg.

Residues analysis of post-harvest imidazole fungicides in citrus fruit by HPLC and GLC.

The determination of imazalil and prochloraz fungicide residues has been carried out by HPLC with an UV detector at 204 nm and by GLC with an electron capture detector (ECD). In both cases fungicide residues were extracted with hexane/acetone (90:10, v/v) after pH adjustment and purified by a liquid-liquid partitioning process. When HPLC was used for prochloraz and imazalil analysis, it was necessary to eliminate the interfering substances with a further clean-up process. This was also required when samples with low residue levels were analyzed by GLC. Recovery was always higher than 70%. The detection limit was 0.04 ppm for the HPLC method and 0.02 for the GLC method. Imazalil and prochloraz residues in "Washington Navel" oranges and "Hernandina" clementine fruits, dipped in a 1000 ppm fungicide solution, are reported.