Carmen Merodio

Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit

Abstract Cherimoya fruit ( Annona cherimola , Mill.) were kept at 20°C in air or in 20% CO 2 for 3 days and then transferred to air, to study the effect of a high CO 2 treatment on phenolic metabolism and ripening-related changes. Total polyphenol levels remained constant while a rapid decline in lignin content was observed in cherimoyas stored in air. However, a sharp increase in PAL activity up to the second day at 20°C was observed. The maximum ethylene production was observed 2 days later. At the end of the CO 2 treatment, ethylene production was inhibited and PAL activity was similar to that found in air-treated fruit. These data suggest that the increase in PAL activity at 20°C was not affected by high CO 2 and does not relate to ethylene. The CO 2 treatment inhibited flesh softening and maintained lignin at levels found in freshly harvested fruit. Exposure to 20% CO 2 also improved internal colour and increased the non-tannin polyphenol fraction, but prevented the decline in the tannin fraction otherwise observed upon ripening in air. We concluded that high CO 2 treatment at 20°C did not enhance PAL activity and lignin deposition although treated fruits retained more lignin after transfer to air. The possible involvement of PAL activity in the supply of important metabolic compounds for early events of ripening will be discussed.

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.

The Relevance of Polyamine Levels in Cherimoya (Annona cherimola Mill.) Fruit Ripening

Summary Free polyamine levels were determined in cherimoya ( Annona cherimola Mill. cv. ) fruit during ripening at 20 °C. Several parameters including respiration rate, ethylene production and total titratable acidity were analyzed. The polyamine prevailing after harvest was spermidine, although a sharp increase was observed in putrescine content during ripening. The possible relationship between this rise in putrescine level and the ripening process was studied on the basis of the pattern of diamine content in fruit stored over a range of low temperatures (10, 8 and 6 °C). Retardation of the ripening process by cold storage was concurrent with a slower rate of increase in the free putrescine level. Moreover, where ripening was inhibited by storage at the chilling temperature (6 °C), no increase in free putrescine was observed. While no variation was observed in spermidine and spermine content in fruit ripening at 20 °C, levels did undergo change during low temperature storage. The possibility that significant accumulation of the free putrescine titer may be associated with high acidity levels in ripening cherimoya tissues is discussed.

Unraveling the roles of CBF1, CBF4 and dehydrin 1 genes in the response of table grapes to high CO2 levels and low temperature

CBFs (C-repeat binding factors) are transcription factors that are rapidly induced by low temperature and that recognize the CRT/DRE element in the promoter of a set of cold regulated genes, the CBF regulon. Dehydrins are proteins that accumulate in plants under stress conditions, such as low temperature, and some form part of the CBF regulon. To investigate their role in the response of table grape clusters to 0°C long storage as well as to 3-day high CO₂ postharvest treatment, we isolated two partial CBF genes (VvcCBF1 and VvcCBF4) and a full-length dehydrin (VvcDHN1a) from Vitis vinifera cv. Cardinal. Hydrophobic cluster analysis (HCA) identified differences in the secondary and tertiary structure between Vitis CBF4s and CBF1s. Overall, our results showed that, in table grapes, the expression of CBF genes is induced mainly in response to CO₂ treatment, suggesting that the response of DHN1a in this fruit could be attributed to a cold-inducible CBF-independent pathway.

Water status and quality improvement in high-CO2 treated table grapes

Unfreezable water (UFW) content in berry tissues (pulp, skin, seed) and rachis of table grape clusters stored at 0°C has been studied using differential scanning calorimetry. The effect of short exposure to high CO2 (20% CO2 for 3days) and the transfer to air were also studied. Water status of pulp tissues was related to the thawing behaviour and the structural characteristics, using low-temperature scanning electron microscopy (LT-SEM). The UFW content in all tissues increased rapidly in response to high CO2 while it remained stable or decreased in untreated clusters. The strong potential of this beneficial gaseous treatment for increasing the UFW content was also evident after transfer to air. The metabolic adjustment caused by exposure to high CO2, which reduced the amount of water available to be frozen, improved stored fruit quality, thus minimising structural damage and reducing water leakage associated with the freezing-thawing process.

Fructo-oligosaccharides in table grapes and response to storage.

Fructo-oligosaccharides (FOS) have been recognized as health food ingredients with a protective effect against environmental stresses in plants. We have analyzed the profiles of individual FOS in Cardinal table grape pulp, until now undetected, and quantified their changes in response to low temperature and high CO2 levels. FOS separation and quantification was carried out using anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and the glucose, fructose and sucrose content of the grapes was also determined. Five FOS were identified and quantified: 1-kestose, neokestose, nystose, nystose b and kestopentaose. While in non-treated table grapes the endogenous FOS remained at steady state levels during storage at 0°C, exposure to 20% CO2 for 3days significant increases the levels of 1-kestose and kestopentaose, members of the inulin series. Considering the competitive advantage afforded by CO2-treated grapes, this transitory FOS accumulation could provide protection against damage caused by low temperature storage.

Characterization of an antifungal and cryoprotective class i chitinase from table grape berries (Vitis vinifera Cv. Cardinal)

Gene expression of a class I chitinase (Vcchit1b) in the skin of table grapes was analyzed as a molecular marker for changes induced at low temperature and also to study the effect of high CO(2) levels modulating transcript levels at 0 degrees C. An active recombinant VcCHIT1b was overexpressed in Escherichia coli, and as the protein was produced as insoluble inclusion bodies, it was solubilized and refolded. The purified recombinant chitinase showed an optimum pH of 6.0 and a temperature of 50 degrees C, retaining activity at 0 and -10 degrees C. Purified chitinase exerted in vitro antifungal activity against Botrytis cinerea. Furthermore, recombinant chitinase was able to cryoprotect lactate dehydrogenase against freeze/thaw inactivation. However, the recombinant VcCHIT1b did not show any antifreeze activity when the thermal hysteresis activity was measured using differential scanning calorimetry.

Regulation of defense and cryoprotective proteins by high levels of CO2 in Annona fruit stored at chilling temperature

This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-beta-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 degrees C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-beta-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO(2) treatment activated a coordinated response of acidic chitinases and 1,3-beta-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO(2)-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO(2) modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo.

Chilling temperature storage induces changes in protein patterns and protease activity in cherimoya fruit

Abstract Storage at 6 °C inhibited the ripening process and caused severe damage in cherimoya fruit ( Annona cherimola Mill. cv. ‘Fino de Jete’). In the present study, we analyzed the modifications in protein pattern, free amino acid content and protease activity of cherimoyas during storage at this chilling temperature. SDS-PAGE analysis revealed non-accumulation of some polypeptides related to the ripening process due to storage at 6 °C, and two-dimensional electrophoresis confirmed the appearance of specific low-temperature polypeptides. While many polypeptides observed in freshly harvested fruit persisted during storage, several acid polypeptides were detected only during the first few days of storage at 6 °C. A substrate-dependent change in protease activity was also found in fruit under chilling temperature storage, as compared to ripening fruit. After a decrease to barely detectable levels during the early phase of cold storage, the proteolytic activity then increased, mainly hydrolizing endogenous proteins to free amino acid components.

Conjugated Polyamine Levels and Putrescine Synthesis in Cherimoya Fruit during Storage at Different Temperatures

Summary In a previous study U. Plant Physiol. 143, 207-212, 1994) we reported a rise in free putrescine levels in cherimoya fruit. ( Annona cberimola Mill.) during ripening. In an attempt to explain such an increase, conjugated polyamine levels, arginine decarboxylase. (ADC, EC 4.1.1.19) and ornithine decarboxylase. (ODC, EC 4.1.1.17) activities were determined in cherimoya fruit stored at 20, 10, 8 and 6 °C. No PCAinsoluble conjugated spermine and PCA-soluble conjugated putrescine titers were detected at any temperature or time interval. PCA-soluble conjugated spermine titers were observed to decline early on in the low-temperature storage period. While PCA-insoluble conjugated spermidine content grew steadily in fruit kept at 20 °C, it was found to decrease throughout the storage period in fruit at lower temperatures. While PCA-insoluble conjugated putrescine levels increased in ripe fruit, they were nonetheless substantially lower than the free-form levels. The rise in putrescine levels in ripening cherimoya fruit was correlated with greatly enhanced ADC activity. No ODC activity was detected under any of the experimental conditions. It is suggested that the ripening-related changes in putrescine levels are regulated via ADC-activity-mediated synthesis and that no conjugation is involved in the process.