María I. Escribano

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.

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.

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.

Increasing Catechin and Procyanindin Accumulation in High-CO2-Treated Fragaria vesca Strawberries

This paper deals with the impact of low temperature and high CO2 levels on flavonols, proanthocyanidins, and anthocyanins, synthesized via branched pathways from common precursors, in strawberries (Fragaria vesca L.). Flavonoids were identified with Q-TOF equipment and quantified by HPLC-quadrupole. Proanthocyanins B1 and B3 accumulated in CO2-treated strawberries, whereas in untreated (air) fruit, flavonoid production was redirected toward anthocyanin accumulation with a sharp decrease in catechin and procyanidin B3 levels. Moreover, in CO2-treated fruit, mainly in those with 20% CO2, anthocyanin accumulation did not decline. Due to its antifungal activity, catechin induction in CO2-treated strawberries could explain the capacity of high CO2 treatments to reduce fungal decay. Ascorbic acid content increased in 40% CO2-treated fruits, whereas in those treated with 20% CO2 an increase in flavonol content was observed. Despite these differences, similar antioxidant capacities were found in untreated and CO2-treated Mara de Bois strawberries.

Ethanolic metabolism in cherimoya fruit during storage at ambient and under high CO2 atmospheres

SummaryCherimoya fruits (Annona cherimola, Mill.) were kept in 20% O2 + 20% CO2 for up to 3 d at 20°C and then transferred to air to study the effect of high CO2 levels on fermentation enzymes, ethanol and acethaldehyde content and ripening evolution. Ethanol and acetaldehyde content increased during ripening in air mainly associated with the first respiration peak. At the end of the short-term high CO2 treatment, cherimoya fruit had a lower aerobic respiration rate while concentrations of acethaldehyde and ethanol were greatly increased compared with those of air-control fruit. The activation of fermentation pathway by high CO2 atmosphere is mainly due to an enhancement in pyruvate decarboxylase (PDC) activity. High CO2 treatment prevented the ripening process but, after transfer to air a decrease in fermentation metabolism was recorded and fruit was able to ripen, showing a typical decrease in tissue pH.

Potent cryoprotective activity of cold and CO2-regulated cherimoya (Annona cherimola) endochitinase.

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO(2)-treated cherimoya fruit (Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a M(r) of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 degrees C, and it retained about 30% of its maximum activity at 5 degrees C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 degrees C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high k(cat) (6.93 s(-1)) value for the fluorogenic substrate 4-MU-(GlcNAc)(3), reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k(cat) at 37 and 5 degrees C, and a low E(a) (11.32 kJ mol(-1)). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze-thaw inactivation of lactate dehydrogenase. The PD(50) was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO(2)-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO(2) concentrations.