Biotechnological control of vegetable ripening and postharvest diseases

Many vegetables exhibit a very short life span after harvesting and require very elaborate measures to expand their life. Reducing the rate of senescence in these crops is not an easy task either by conventional or biotechnological methods. The main obstacle to devising new technologies is the complexity of the problem and lack of basic knowledge about the biochemical and cellular processes accompanying post-harvest induced senescence. This is accentuated by the extraordinary variety of tissue types that are commercialised. Early attempts to use genetic manipulation to alter senescence have been based on hormone physiology, either enhancing cytokinin production or blocking ethylene production or perception.

In order to extend the post-harvest life of leafy vegetables we first need to focus on the events that occur in regular leaves during senescence. It has been known for some time that cytokinins can delay leaf senescence and that during senescence there is a drop in endogenous cytokinin levels (van Staden et al., 1988). Overproduction of IPT, a bacterial enzyme that catalyses the rate-limiting reaction in the biosynthesis of cytokinins under the control of the strong constitutive promoter CaMV 35S, resulted in transgenic plants with high levels of cytokinins and delayed leaf senescence. But these plants also showed many developmental abnormalities since apart from senescence cytokinins are implied in a myriad of other developmental processes (Smart et al., 1991). The last example stresses the importance of the availability of adequate promoters to express the right gene in the right place at the right time. An ingenious solution to the use of cytokinins to delay senescence has been provided by Gan and Amasino (1995) who placed the IPT gene under the control of SAG12, a senescence-specific promoter. In this system, the onset of senescence activates the SAG12 promoter, leading to the production of cytokinins. The accumulation of cytokinins inhibits the emerging senescence process and consequently reduces the activity of the SAG12 promoter, therefore avoiding the accumulation of cytokinins. Transgenic tobacco plants obtained in this way contained leaves with extremely delayed senescence that maintained high levels of photosynthetic activity. It remains to be proved whether this approach can be applied to leafy vegetables.

Ethylene often has an opposite effect to cytokinins in promoting senescence but its role is not likely to be essential for the regulation of the process in vegetative plant tissues. However, blocking the production or the perception of ethylene could have a positive effect in the longevity of green tissues. Transgenic tomato plants with reduced levels of ethylene production have shown retarded leaf senescence (John et al., 1995). Transgenic Arabidopsis plants with the etr1-1 dominant mutation that renders them insensitive to ethylene have also shown delayed leave senescence (Grbic and Bleecker, 1995). It is important to remark that in both cases delay was observed in the onset of senescence but once the process had been started it proceeded at normal speed. From the available data it seems that senescence-related genes are activated by ethylene only if the leaf is ready to senesce; when that happens ethylene enhances the process.

In floral vegetables such as broccoli, ethylene is likely to play an important role in both the onset and the regulation of the senescence process. Ethylene has already been proven to play such a role in flowers such as carnations (Woodson et al., 1992). Transgenic broccoli has been produced containing antisense copies of a tomato ACO gene (Henzi et al., 1999a; Henzi et al., 1999b). Analysis of respiration rates, ethylene production and ACO activity performed in several transgenic lines showed puzzling results. Transgenic lines showed a marked increase in ethylene production in the early phase of post-harvest with levels three times higher than control samples; nevertheless, 74 hours after harvest ethylene production in controls markedly increased whereas the transgenic lines showed reduced ethylene levels. Respiration rates in control and transgenic samples were comparable immediately after harvest but transgenic samples showed a linear decrease of respiration up to 98h after sampling. Paradoxically, ACC oxidase activity levels in the transgenic samples were always higher than controls. In order to evaluate and interpret these experiments further research is needed to determine the gene expression patterns of the endogenous ACO genes since the authors used a relatively low homology tomato ACO gene in their genetic constructs. Preliminary agronomic evaluation has revealed some promising transgenic lines with significant improvements over the controls (Henzi et al., 2000).

In addition to the factors discussed above, some vegetables such as lettuce, broccoli, cauliflower and asparagus are harvested while they are still immature and undergoing a phase of rapid growth in the plant. In these vegetables there are very rapid changes after harvest with broccoli losing large amounts of sucrose within four hours of harvesting. Significant changes in gene expression are also observed with accumulation of different transcripts such as asparagine synthase that catalyses the synthesis of the amino acid asparagine. The metabolic parameters of immature vegetables after harvest undergo important changes with loss of proteins and lipids and accumulation of free amino acids and ammonia that ultimately lead to tissue breakdown. This data strongly resembles starvation responses and suggests that starvation might be a critical stress regulating the senescence process in harvested immature vegetables.