Control of fruit firmness

Softening is an important contributor to losses experienced during the handling and transport of fruit. Among the genes involved in firmness, the most extensively studied is the one coding for polygalacturonase (PG) (Della-Penna et al., 1986; Grierson et al., 1986), a cell wall enzyme that catalyses the hydrolysis of polygalacturonic acid chains. Polygalacturonic acid is an important component of the plant cell wall that significantly contributes to the fruit firmness. Partial silencing of the PG gene has been achieved in tomato by sense and antisense techniques. Experiments using either a partial or the full length PG gene successfully reduced the levels of PG mRNA and enzyme activity (She ehy et al., 1988; Smith et al., 1988; Smith et al., 1990). It is important to remark that these were the first examples of the successful use of the antisense technique in plants. Different transgenic lines showed different degrees of gene silencing, indicating that the position where the transgene is inserted in the genome plays an important role in the effectiveness of the technique, as has been emphasised in Tools of genetic engineering in plants. In tomato, PG has been extensively associated with softening because of the temporal and spatial coincidence of the increase of PG activity during the softening period of the fruit. Contrary to all expectations, the antisense PG fruits produced by Smith et al., (1988) did not show any appreciable change in softening when measured by classical methods (such as compression tests). A debate was started on whether PG had any effect in internal softening of the fruits, and the widely accepted idea that PG was directly responsible for the softening process was shaken. Nevertheless, a new and more detailed study of transgenic sense and antisense PG plants revealed a number of important changes in the transgenic fruits (Schuch et al., 1991). Low PG tomatoes were more resistant to cracking and splitting than regular fruit. They also had superior handling and transport characteristics showing a severely reduced degree of damage during those processes (Schuch et al., 1991).

How much inhibition of the PG gene is necessary to observe any changes in the fruit phenotype? This factor has not been fully answered yet because of the difficulty in regulating the exact amount of gene silencing in transgenic lines. Genetic engineering of plants has not reached the high level of sophistication needed to pre-determine or precisely regulate the level of gene silencing. Nevertheless, molecular analysis of the transgenic tomato lines showed that fruits in which PG activity had decreased to less than 1% of normal levels contain longer polygalacturonic acid chains, affecting cell adhesion and making the fruits sturdier.

Agronomically, the effect of low PG can be translated in fruits that can be left on the vine for a longer time, therefore enhancing the flavour, since the softening process has been slightly delayed. Interestingly, the main commercial use of the low PG tomato fruits has been in the processing industry. Transgenic low PG tomatoes show enhanced viscosity of the processed products and produce less waste. The new characteristics of these fruits have also allowed us to simplify the manufacturing process.

‘Flavr Savr’, the commercial name for a low PG tomato, marked an important milestone in plant biotechnology being the first genetically modified plant food to reach the market, commercialised by Calgene in the USA in 1994. Zeneca and associates are currently commercialising a tomato puree based on genetically modified low PG tomatoes. This product went on sale in the UK in 1996.

The results of the genetically modified low PG tomatoes have shown that although PG plays a significant role in texture changes during fruit ripening in tomato, it is not the primary factor controlling softening. There are a number of cell wall modifying enzymes that have been characterised at the biochemical level and shown to be active during fruit softening including cellulases, pectinesterases, galactanases, etc. It is also important to remark that, based on the research data available, it is likely that there is not a single softening pathway common to all fruits. Different species have been shown to have very different cell wall modifying enzymes’ activity patterns during ripening, therefore it is not possible to devise a single universal strategy to control softening.