Control of ethylene synthesis and perception

Ethylene is one of the simplest organic molecules with biological activity and is the only gaseous hormone known to date. In climacteric fruits ethylene controls the onset and rate of ripening and therefore several strategies have been devised to interfere with either the rate of ethylene synthesis or its perception by the fruit. The elucidation of the ethylene biosynthetic pathway by Yang and coworkers(1984) (Fig. 5.1) openedthe door for the isolation of the enzymes involved and the cloning of the corresponding genes.

Aminocyclopropane carboxylate (ACC) synthase and ACC oxidase are the key enzymes in this pathway controlling the last two steps in the production ethylene. Both of them are encoded by multigene families and normally only one or two members of the family are active in the fruit during ripening. In tomato, the ACC synthase gene active during ripening (LEACC2) was silenced using antisense techniques effectively reducing the production of ethylene by the ripening fruit by 99.5% (Oeller et al., 1991). While control fruits begin to produce ethylene 48–50 days after pollination and immediately undergo a respiratory burst, genetically modified tomatoes produced minimal levels of ethylene and failed to produce the respiratory burst (at least during the 95-day period analysed in the report). Transgenic fruits started showing symptoms of chlorophyll degradation 10 to 20 days after the control fruits turned to yellow, and eventually developed an orange colour two months later; meanwhile control tomato fruits needed only ten days for the transition from full mature green to fully ripe red tomatoes.

The transgenic tomatoes studied by Oeller et al., never turned red and soft and never developed aroma when kept in the plant or stored in an air atmosphere. Obviously, these characteristics are not desirable for a commercial fruit crop since the consumer wants a ripe product with all the attributes of colour, aroma, flavour, etc., fully developed. An obvious question arises of whether this phenotype is reversible by treatment with ethylene or the genetic change has created fruits completely unable to undergo the ripening process. When mature green transgenic fruits (49 days after pollination) were treated with ethylene, they developed a fully ripe phenotype within seven days (as opposed to two days for control fruits). The ethylene-treated genetically modified ripe fruits were indistinguishable from naturally ripened control fruits in colour, texture, aroma and compressibility. Although scientifically this work is of great importance, such extreme phenotypes may not prove useful in a commercial situation and intermediate phenotypes should be targeted. The above studies strongly suggest that ethylene is the trigger that starts the respiratory burst in climacteric fruits and controls the rate of ripening.

The ripening-related ACC oxidase gene has also been cloned in tomato and its expression inhibited by 95% (Hamilton et al., 1990; Picton et al., 1993). This level of inhibition did not block ripening in the transgenic plants allowing normal development of the fruits but delaying the onset of senescence, over ripening, cracking of fruits and other general over-ripening effects. Nevertheless, when mature green fruits were picked from the plant they never fully ripened. Even when exposed to ethylene, although they developed full red colour, the levels of carotenoids never reached those achieved by plant-grown fruits (Pictonet al., 1993).

Instead of altering the levels of enzymes controlling the biosynthesis of ethylene, two commercial companies (Monsanto and Agritope) have opted for alternative strategies aimed at depleting the intermediate substrates of the pathway. Monsanto used a bacterial enzyme (ACC deaminase) to drain the cell of the immediate precursor of ethylene (ACC). Overexpression of an ACC deaminase gene in tomato plants led to a marked depletion of the levels of ACC and therefore reduced the availability of this precursor to be converted into ethylene (Klee et al., 1991). Transgenic plants overexpressing ACC deaminase were indistinguishable from controls with no differences observed during development even though there was a dramatic decrease in the levels of ethylene produced in vegetative tissues. Out of all the independent transgenic lines obtained, the best one produced fruits with ethylene levels of only 10% of the controls. When fruits were picked from the plant at the breaker stage and stored at room temperature, controls achieved fully red stage in seven days compared with 24 days for the transgenic fruits. Softening behaviour was also affected with controls showing a strong incidence of softening two weeks after picking; in contrast transgenic fruits remained firm for five months. When fruits were left on the plant to ripen, transgenic fruits remained firm for much longer than controls and did not abscise for more than 40 days. Agritope has used a bacteriophage gene encoding S-adenosyl methionine (SAM) hydrolase, in conjunction with a ripening specific promoter, to hydrolyse the first intermediate of the ethylene biosynthetic pathway (SAM) in ripening cherry tomato fruits (Kramer et al., 1997). The transgenic fruits exhibited a delayed ripening phenotype and a reduction of spoilage due to over-ripening.

Is it possible to control ripening in other fruits? All the studies previously described have been achieved in tomato. The reason for the choice of this system is clear: tomato is a very important crop with an extensive research history into the biochemistry and genetics of ripening. In addition tomato transformation is relatively easy when compared to other fruit species and the results of a transformation experiment can be evaluated in a glasshouse in a year (as opposed to an entire field and 5–7 years for fruit trees). Nevertheless there are clear indications that the approaches described earlier can be applied to other crops as is the case of melon. Ayub et al. (1996) used antisense techniques to inhibit ACC oxidase levels and concomitant ethylene production during ripening of cantaloupe ‘Chanterais’ melons. This variety has excellent eating quality but a notoriously poor storage capacity. Genetically modified plants were produced with ethylene synthesis severely impaired (less than 1% of controls). Storage capacity was extended, with transgenic fruits remaining fresh after ten days at 25°C while control fruits had spoiled. The softening of the fruits was also affected with transgenic fruits remaining twice as firm as non-transformed controls. Exposing the transgenic fruits to external ethylene restored the ripening phenotype. A recent report by Ben-Amor et al. (1999) has revealed that the lowethylene melons have considerably less sensitivity to chilling injury. This is an additional important improvement since most tropical and subtropical fruits are very sensitive to low temperatures and this fact severely impairs their transport and storage potential causing significant losses. The antisense ACC oxidase melons did not develop chilling injury when stored for up to three weeks at 2°C while controls exhibited extensive damage.

An alternative to the control of ethylene production during ripening is to decrease the sensitivity of the fruit to the hormone. It has been established that during ripening, fruits not only increase the production of ethylene but they also become more sensitive to it (Theologis, 1994). The cloning of the ethylene receptor (etr1) has opened the door to the manipulation of ethylene perception instead of ethylene production (Chang et al., 1993). A mutated version of etr1 in Arabidopsis (etr1-1) confers ethylene insensitivity as a genetically inheritable dominant trait. The same mutated gene has also been introduced into tomato and petunia conferring ethylene insensitivity and producing fruits that fail to ripen or flowers with extremely delayed senescence respectively (Wilkinson et al., 1997). It is clear that complete ethylene insensitivity is not a desirable trait for a fruit since it would render the fruit unable to ripen even when exposed to ethylene. On the other hand, selective, partial or induced insensitivity to ethylene could be commercially useful.