AGRE-0015 Improvement of Storage Capabilities for Fresh Fruits Through the Biotechnological and Biotechnical Control of Ethylene

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Biotechnology : ECLAIR Cluster VI - Production and Storage : Storage/Transport

Ethylene

CURRENT STATE OF THE PROJECT

The improvement of the ripening characteristics of melon by genetic transformation is one of the central themes.

Ethyleneforming enzyme (EFE)related clones were isolated from the melon cDNA library among which 2 have been completely sequenced, pMELI and pMEL2. A melon genomic library was generated in EMBL3 vector containing 2.1 x 10^6 independent clones amplified to 1010. This work is firmly in the core of the project and the cDNA and genomic libraries have been given to other partners. Among the 6 calmodulinrelated clones isolated, one has been fully sequenced. A 75 kD polypeptide corresponding to the Calcium channel protein has been partially purified.

Genetic transformation of melon necessitates gene transfer. Transient expression of the CAT gene was obtained in melon protoplasts. Regeneration of melon was improved through the inhibition of ethylene action. Several transgenic plants were obtained and chimeric DNA inserted into the genome was stably transferred to progenies Tl and T2.

In vitro EFE activity of melon was successfully stabilized and partially purified. EFE recombinant protein has been produced and polyclonal antibodies have been raised.

The physiology of the ripening of melon is affected by the enzymes of ethylene biosynthesis. EFE activity increases sharply 4 days before harvest. It was found also that ABA content of the fruit increases with maturity. Galactopyranosidase, arabinopyranosidase and glucopyranosidase were found to be largely responsible for softening of melon fruit.

The effect of chilling on cell membranes was studied in tomato. A detailed study of the pattern of plasma membrane proteins during ripening was performed. Changes in tissue texture and water mobility were visualised using NMRimaging for nondestructive monitoring of ripening in tomato fruits.

Ripening and other senescence changes could be slowed if the action of ethylene was inhibited. Some of newly synthesis Ed furyl acrylate and thiophene derivatives can delay tomato ripening. A gaseous ethylene antagonist has been shown to reduce ethylene production and respiration. Maintaining ethylene at low level by carbon dioxide shock treatments delayed the softening of tomato and kiwi fruit.

The storability of fresh fruit can be extended by creating modified or controlled atmospheres. Film packaging is a potential mean to modify the atmosphere surrounding the fruits. A laser perforating process has been tested giving encouraging results but too expensive for practical application. Reducing the ethylene level with a pilotscale reactor in the storage room containing Granny Smith apples reduced the incidence of scald. The respiratory rates of nonclimacteric fruits under film packaging have been determined. Plastic film packaging of melon fruits reduced weight loss and development of skin colour at early stage of ripening without effect on flesh softening. In tomatoes picked at the right ripening stage, it slowed down colour and ripening changes. Other experiments are also in progress to protect fruits against scald by mixing edible fruit coating with foodedible antioxidants.

The improvement of the ripening characteristics of melon by genetic transformation is a central theme in the present project. It is therefore particularly pleasing to report that work in this area is ahead of schedule. Professor Gricrson's group (C. Watson, A. Turner and S. Picton) at Nottingham and Dr Balague at INP ENSA Toulouse have purified and characterised an active poly A+ mRNA fraction from preclimacteric and ripening melons (supplied by TEZIER), synthesis Ed cDNA from the poly+mRNA and prepared doublestranded cDNA, and cloned the cDNA to generate a ripening specific cDNA library from melon.

It was demonstrated at Nottingham that the pTOM13 cDNA isolated from tomato fruit was encoding EFE. This clone was then used as a probe to isolated EFErelated clones from the melon cDNA library. Sixteen clones have been isolated, among which 2 have been completely sequenced, pMELI and pMEL2. A melon genomic library was generated in EMBL3 vector containing 2.1 x 10^6 independent clones amplified to 10^10). The rapid completion of this work owes much to exchange of personnel between Nottingham and Toulouse. Indeed this work is firmly in the core of the project: the cDNA and genomic libraries have been given to other partners.

Professor Trewavas and Dr Oliver (Edinburgh) have screened the cDNA library for calmodulin clones. Among the 6 calmodulinrelated clones isolated, one has been selected and sequenced. It has 82% homology with a potato cDNA probe.

Calcium is also the theme of Dr Ranjeva's work at UPS, Toulouse but in this case attention is focused on the channel which permits calcium to cross the plasma membrane. Maintenance of a calcium gradient across this membrane is likely to be important in maintaining good storage capability and avoiding chilling injury. A 75 kD polypeptide has been partially purified after photo affinity labelling with the calcium channel blocker, LU 49888. The channel is located at the plasma membrane, and reconstruction of a functional calcium channel has been achieved by insertion into proteoliposomes.

Genetic transformation of melon will necessitate gene transfer. Already, Dr Alibert at INPENSA, TOULOUSE has obtained transient expression of the CAT gene in protoplasts of melon under the control of the 35S promoter of the CaMV. Constructs containing CAT and EFE (pTOM13) cDNA were introduced via electroporation into protoplasts. These were regenerated into green nodules and roots, but so far entire plant lets have not been obtained. Dr S. Bentahar at BIOCEM has reported on the optimisation of the conditions for regenerating plant lets from cotyledons, and have succeeded in bringing to flowering plants of the Vedrantal variety, transformed via Agrobacterium to express the GUS gene. The most efficient bacterial strains were determined. Using kanamycin selection, several transgenic plants were obtained and the chimeric DNA inserted into the genome was stably transferred to progenies Tl and T2.

In the work of Drs Fallot and Roustan at ENSA, Toulouse regeneration of melon plants was found to be inhibited by ethylene, but could be improved by using the inhibitor of ethylene action, silver nitrate.

Ethylene production in fruits is regulated by the activity of two enzymes: ACC Synthase and the ethyleneforming enzyme (EFE). Dr Pech's Laboratory (A. Latche and M. Bouzayen) at INPENSA Toulouse, partially purified the ACC synthase from ripening melons, but with publication of the sequence of the tomato enzyme from another laboratory there was a change of direction and this laboratory focused on the preparation of cDNA by PCR technology (J.M. Lelicvre). Dr John's Laboratory (P. Ververidis and J.J. Smith) at READING succeeded in stabilising in vitro the EFE activity of melons. This is the first time that the activity of this enzyme has been studied in vitro from any plant. An important clue to the method for stabilisation came from previous work from Professor Grierson's Laboratory at NOTTINGHAM. For many years, it has been believed that the EFEwas a membranebound enzyme, but the recent findings from READING show that it is a perfectly soluble enzyme, which enabled it to be partially purified and characterised in terms of its kinetics and inhibitor sensitivity. Unusually for a dioxygenase enzyme, activity was independent of the presence of 2-oxoglutarate. EFE recombinant protein has been produced by INPENSA TOULOUSE (J.M. Lelievre) and polyclonal antibodies have been raised.

The way in which the activity of the enzymes of ethylene biosynthesis affect the physiology of the ripening melon has been studied by Dr Vendrell's Laboratory at CSIC, Barcelona, who have found that EFE activity increases sharply 4 days before the commercial harvest date, but while EFE activity decreases after the climacteric, this is not so with ACC synthase, so that ACC (and MACC) continue to increase during the climacteric. It has been found that the ABA content of the fruit increases with maturity and with the development of ripening. In conjunction with GIRONA FRUITS and COOP. de Lerida, Dr Vendrell's Laboratory is carrying out a longterm study of the effect of ethylene removal and treatments including edible coatings supplied by SURFACE SYSTEMS INTERNATIONAL (SEMPERFRESH), Reading. Some important field factors that affect fruit quality have already been identified.

One of the most changes during fruit ripening is the softening triggered by ethylene. Drs Buret and FilsLycaon at INRA, Avignon working in conjunction with Dr Hobson, Littlehampton have identified a galactopyranosidase, arabinopyranosidase and glucopyranosidase as being largely responsible for softening in melon fruit, and have found that the ß-galactosidase and the Aarabinopyranosidase activities are due to the same enzyme, which has been partially purified.

For commercialisation the extension of shelflife demands that fruit organoleptic quality be maintained, so that Drs Kanellis of IMBB, HERAKLION and Dourtoglou of VIORYL, Athens have made an important contribution to the definition of fruit quality in melon, by identifying the volatiles which provide melon with its characteristic aroma. Sulfo compounds make an important contribution, and these are derived by myrosinase action from glucosinolates. Two enzymes involved in the production of aromas have been studied: PAL and myrosinase. PAL activity is higher in young fruits and increases during wounding as a consequence of a stimulation of mRNA synthesis. Myrosinase has been purified to quasihomogeneity, and its activity has also been found to be higher in immature fruits. These results provide the basis for a detailed understanding of their participation in the production of volatiles in the melon.

This foregoing basic research has been done with melons as a model system, because this is a rapidly ripening climacteric fruit, and transgenic plants can be raised. It was also of enormous commercial interest to control melon ripening. However to widen the applications of the present project other fruits have been examined.

Drs Soudain and Nguyen at CNRS MEUDON have studied the effect of chilling on cell membranes in the tomato. The breakdown of monogalactosyl diglycerol has been found to be a good marker for the induction of chilling. The plasma membrane, generally considered to be more sensitive to chilling injury, has been purified by phase partition and freeflow electrophoresis, and characterised by marker enzymes and lipid composition. A detailed study of the pattern of plasma membrane proteins during ripening was performed.

Dr Pradere at UPS, Toulouse has established the feasibility of using NMRimaging for nondestructive monitoring of ripening in tomato fruits, in which it has been possible to visualise the changes in tissue texture and water mobility. The spectra obtained by localised spectrometry show that it is possible to follow with this nondestructive method changes in sugars, organic acids, etc.

Ripening and other senescence changes could be slowed if the action of ethylene was inhibited. Dr Ambrosi at CFPI has found that some newly synthesis Ed furyl acrylate and thiophene derivatives were active ethylene antagonists in model systems. Some of these compounds used at 300 ppm can delay tomato ripening by several days and are therefore of possible practical use.

A gaseous ethylene antagonist has been investigated by D. Fath at L'AIR LIOUIDE, who has compared its effectiveness to known treatments such as carbon dioxide shocks. This compound shows great promise, as it can delay the ethylene peak, reduce ethylene production, and reduce respiration (Dr Soudain, CNRS MEUDON) while fruit quality is retained. Large scale tests made in cooperation with wholesale distributors (MM Aubin and Dalle, POMONA) have revealed the potential of the compound commercially.

Intermittent carbon dioxide shock treatments have been shown to delay the softening of kiwi fruits and tomato (Drs Nicolas and Rothan, INRA, Avignon) by maintaining ethylene production at a low level. After transfer to air, residual effects were observed in relation with lower ethylene production and a reduction of EFE activity.

The storability of fresh fruits can be extended by creating modified or controlled atmospheres. Film packaging is a potential means to modify the atmosphere surrounding the fruits. Dr Torre's group at INPENSC TOULOUSE has devised an experimental system allowing one to determine the permeability of film gases under controlled films or membranes as prepared by RHONE POULENC (Dr Corsi). of the methods which have been examined for microperforation, ultrasonic heat source gave insufficient control of permeability, but a laser perforating process has been tested and gave encouraging results, giving holes below 15 m diameter.

This system lends itself to application in the fruit packing houses, where the perforations applied could be modified so that the film was closely adapted to the requirements of each particular commodity being enclosed by the film. For the present the method is technically feasible, but it appears to be too expensive for practical application. However technical improvements, like the use of holographic mirrors, are being explored which could reduce costs.

The elimination of ethylene which accumulates in the storage rooms can result in an extension of the shelflife of fresh fruits and a lowering of storage disorders. The work undertaken by Dr Torre's group at INPENSC TOULOUSE has consisted in setting up a laboratory reactor capable of oxidising ethylene at low temperatures (100-120°C). A pilotscale reactor has been used at an experimental fruit station for removing ethylene from a CA store of Granny Smith apples (2.5% oxygen). Compared to a untreated room with 70 ppm ethylene, the reactor reduced the ethylene level down to less than 3ppm. Removal of ethylene reduced the incidence of scald, but to a less extent than when the oxygen level was reduced to about 1%.

Studies on the behaviour of nonclimacteric fruits (oranges, mandarin and pepper) under film packaging have been initiated by Dr Romojaro's group in MURCIA. The respiratory rates of whole fruits or parts of them have been determined for various temperatures. The conditions for chemical peeling of oranges have been also studied with the aim of preparing readytouse (minimally processed) fruits under plastic films.

For the industrial simulation of the new techniques, some large scale experiments had been undertaken in the first year, however it was in the second year that this work really took off. In one experiment carried out by Dr Baccaunaud (AGROTEC) involving 600 kg of melons, the effect of plastic film packaging was examined. The technique greatly reduced weight loss and the development of skin colour (early stages of ripening only), but there was no effect on flesh softening. A similar experiment with tomatoes showed that film packaging slowed colour changes and softening when applied at the right ripening stage.

Previous trials had suggested that the effectiveness of the edible fruit coating, Semperfresh, as protection against scald could be enhanced by mixing it with food compatible antioxidants. Under the supervision of SURFACE SYSTEMS INTERNATIONAL (SEMPERFRESH), a large scale trial of a variety of formulations has been started, replicated at the laboratories of two partners: LERIDA (Dr Recasens) and READING (Dr A.D. Bauchot). Each trial involves the treatment of about 1 tonne of Granny Smith apples. The results will become available during spring of 1992.

CONCLUSION

During the first year or so of this project, disappointing progress was made in some areas due to changes in personnel and to economic difficulties of some industrial partners. However, it is apparent from the above progress report that these difficulties have now been overcome, and progress has been made in all areas. Even though this is only the midterm of the project, the partners at the commercial end of the distribution chain have already become involved. Besides the obvious scientific advances that have been made in this project, several potential precompetitive products have been developed all capable of controlling ethylene: specific genes, new gases, coating formulations and plastic film techniques. In addition commercial links have been generated by the project, for example CFPI/SURFACE SYSTEMS INTERNATIONAL (SEMPERFRESH). An essential contribution to the success of the project has been the interchange of scientists between the partner laboratories. This has been instrumental in the preparation of new proposals for collaborative work.