Tomatoes are one of the largest vegetable crops in the US. While we are most familiar with fresh tomatoes, they comprise a relatively small share of the market. Most tomatoes are used for processing - canning, ketchup, paste, sauce, pizza sauce, etc. An excellent source of background information on tomatoes can be obtained from David Rhodes' webpage for HORT 410
The tomato has been one of the first targets of plant biotechnology for a number of reasons. First, tomatoes are in the same family, the Solanaceae, as tobacco. Tobacco was widely used as a model for plant transformation, so it was fairly easy to develop transformation systems for tomato. Second, there are a number of characteristics of fresh market tomatoes that consumers are unhappy about: quality, flavor, shelf life. Most of the year, fresh tomatoes bought in the store are of very poor quality and have little resemblance to fresh garden tomatoes. Third, processing of tomatoes is an expensive, energy consuming task, and processors are interested in finding ways to cut costs, improve the product, and develop new products.
Modification and improvement of tomato by traditional breeding methods has been going on for many years. The tomato originates in South America and there are many collections of germplasm made from this region. In addition, there are several related species which can be crossed to tomato. These related species have been used as sources of genes for improvement of tomatoes by breeding. Genes for resistance to many diseases have been identified in the wild relatives (such as Lycopersicon pimpinellifolium) and then introduced into normal tomato (Lycopersicon esculentum) in a traditional backcross breeding program. This takes many years to complete but is an effective and successful method to add new genetic features to tomato, as well as other crop plants. Genes have also been identified that slow down or arrest the ripening process. A number of these mutants that are unable or slow to ripen (rin ripening inhibited, and nor never ripen) were identified and studied by a former professor in the Purdue Horticulture department, Ed Tigchelaar. Attempts have been made to use these genes to develop slow or delayed ripening tomatoes. However, it is my understanding that efforts to develop tomatoes with improved ripening properties by using these genes have not yet been successful. Most of the tomatoes remained too firm to be eaten or processed.
Before we can think about how to modify tomato ripening, we must first have an understanding of what occurs during ripening. This is the penultimate stage in fruit development.
After fertilization, the tomato fruit grows, first through cell division and then by cell expansion, until it reaches its maximum size. This takes about 40 to 50 days. The mature green fruit then undergoes a number of dramatic changes: increase in ethylene production, increased respiration, synthesis of red pigments (lycopene), softening of the fruit, development of flavors, conversion of starches to sugars, etc.
All of these processes are regulated. Ripening is not just the random deterioration of biological processes in the fruit. (The mutants that fail to ripen properly are proof of this. The action of certain genes is required for normal fruit ripening; when those genes are inactive, as a result of a mutation, ripening does not occur.) Specific biochemical processes are activated during fruit ripening that account for the changes that are collectively known as ripening.
It is now known that the plant hormone ethylene plays a central role in tomato fruit ripening. When synthesis of ethylene is blocked, using chemical inhibitors of the enzymes of ethylene synthesis, tomato fruits will not ripen.
The pathway of ethylene biosynthesis is as follows:
SAM is S-adenosyl methionine, ACC is aminocyclopropane-1-carboxylic acid. The enzyme that catalyzes the first step is ACC synthase, and the second step is catalyzed by ACC oxidase.
Ethylene then acts as a hormone to regulate the ripening process. Ethylene is also involved in other facets of plant growth, including seedl germination and seedling growth, leaf abscission, petal senescence, responses to environmental stress. In fruit ripening, ethylene activates the expression of many genes. These gene products, usually proteins, are then responsible for the specific changes that occur during ripening.
It is clear that there are a number of steps at which the biotechnologist could intervene to modify fruit ripening, as indicated below. These strategies include:
The first method to reduce the synthesis of ethylene that I will describe has been developed by a relatively small biotech company in Oregon called Agritope. The principle they have used is one that I like to call "metabolic interference", where a substrate in a metabolic pathway that will normally be used to synthesise a specific compound (X) is diverted to something else, thereby preventing the synthesis of compound X. Agritope has developed a method to prevent SAM from being converted to ACC, thereby blocking or reducing the synthesis of ethylene. This has been accomplished by expressing a gene that encodes SAM hydrolase. This enzyme converts SAM to non-toxic byproducts (I can't tell you what just now!) that are recycled within the plant cell. The most important point is that SAM is not used to make ACC. The gene encoding SAM hydrolase was originally cloned from a bacterial virus.
To express this enzyme in tomato fruit and prevent them from ripening, the gene from the bacterial virus first had to be modified so that SAM hydrolase would be expressed in the correct place and time - the mature green fruit before or just as it would normally start to ripen - to prevent synthesis of ethylene in fruit. The modules that are required for this chimeric gene are:
Transgenic tomato plants containing this gene have been produced by Agrobacterium-mediated transformation, and the gene is expressed appropriately. I will discuss how the fruit ripen (or not) in all of these plants that have been modified to have low ethylene production in the fruit at the end of this section.
The second method is another example of metabolic interference which has been developed by Monsanto and is very similar to that described above. However, instead of diverting the first compound in the ethylene biosynthesis pathway, SAM, this approach prevents ACC from being converted to ethylene by ACC synthase. Monsanto scientists discovered a bacterium that was able to metabolise ACC. They showed that an enzyme called ACC deaminase was resbonsible for this metabolism of ACC, and cloned the gene for this enzyme from the bacterium. As described for SAM decarboxylase, this gene from the bacterium must be manipulated so that it can be expressed in plants. The assembled chimeric gene requires:
Again, transgenic plants were produced that expressed the ACC deaminase, and some were obtained that had reduced ethylene production. These will be discussed below.
A different strategy would be to look for mutants that are defective in ethylene biosynthesis. These could arise in a number of ways, such as a mutation in one of the genes for ethylene synthesis, but there are other possibilities as well. However, I am not aware of mutants being identified that are deficient in ethylene synthesis in crop plants.
An alternative approach would be to use molecular techniques to inactivate one of the ethylene biosynthesis genes. In bacteria, many yeasts, and even in mammals such as mice, methods have been developed to make specific genetic changes, such as removing a single gene from the genome. These rely on homologous recombination to replace genes in the chromosome. I am sure these methods will be developed in the future for plants, but at present they are not available.
In the absence of such methods to remove specific genes in plants, two alternative approaches have been developed to reduce or eliminate the expression of individual plant genes. These are known as antisense RNA, and gene silencing (also known as co-suppression). To my knowledge, gene silencing (co-suppression) as used in plants is not used outside plants to manipulate gene expression. However, the use of antisense RNA and related strategies to inactivate specific genes is being developed and evaluated for medical use where there are some conditions that result from activation of specific genes. If expression of these genes could be switched off, it might provide a treatment for some diseases, including cancers. Antisense RNA is a widely used technique in plants, not just to alter tomato fruit ripening, so it is important that we understand the principles behind this approach.
Before we look at how antisense RNA is made and works, let's review some of the critical features of the RNA that is used to make proteins, messenger RNA (mRNA).
- mRNA is a single stranded nucleic acid molecule
- the sequence of bases in the RNA is the same as in one strand of the DNA from which it was transcribed, except that the base U replaces T
- this RNA serves as the template to make a specific protein, by translation on ribosomes
If the RNA is not translated into protein, then expression of that gene will be effectively blocked. This is what antisense RNA is designed to accomplish.
How can a plant be manipulated to produce antisense RNA? Transgenic plants have to be made containing a new chimeric gene which will make antisense RNA when it is transcribed. In a normal gene, the promoter directs RNA polymerase to transcribe mRNA, using the lower strand of the DNA as template to make an RNA that has the same sequence as the upper strand of the DNA, with U in place of T. The transgene used to make antisense RNA makes a transcript of the same piece of DNA, but instead of using the bottom strand as template, it uses the upper strand and is transcribed in the opposite direction. The result is that the RNA transcribed form this "antisense RNA gene" has the same sequence as the lower strand.
The RNAs transcribed from the normal gene (the mRNA or sense RNA) and the antisense gene (antisense RNA) are present within the same cell. As with DNA strands that have complementary sequences, the sense and antisense RNAs will form double strands by hydrogen bonding between the bases on the two strands. This double stranded RNA cannot be translated into protein.
This is the basis for using antisense RNA to inactivate the expression of specific genes in plants. Once these double stranded RNA molecules are formed, it appears they are rapidly degraded by some unkown mechanism within the plant cell.
How has this approach been used to block ethylene synthesis in tomato fruits? The genes encoding the enzymes of ethylene biosynthesis, ACC synthase and ACC oxidase have been cloned from several species. As with many other genes, once the first gene has been cloned in one species, it is normally quite easy to isolate the gene encoding the same enzyme from other species. These genes have formed the basis for making antisense RNA for either of these two genes that are required for ethylene biosynthesis. The essential components of the chimeric genes used to express these antisense RNAs are as follows:
Transgenic tomato plants have been produced that express antisense RNA for ACC synthase (first by researchers working for the USDA in California) and ACC oxidase (first done by researchers in England in collaboration with Zeneca, experiments that serendipitously led to the identification of the gene for what was until then a somewhat mysterious and elusive enzyme known as the ethylene forming enzyme). In the "best" transgenic plants expressing antisense RNA for either of these genes, a small fraction of the plants had essentially no ethylene production in the fruit. These fruit were then shown to be unable to ripen unless they were continuously treated with ethylene. These experiments were then critical in confirming the role of ethylene in ripening.
The second strategy used to make transgenic plants with a gene that blocks synthesis of ethylene is called gene silerncing or co-suppression. This was discovered when people were doing some of the earliest experiments with transformation of plants, trying to find out what would happen if you transformed plants with a gene for an enzyme in the pathway for making anthocyanin pigments in flowers. When this gene was put into plants that already produced these pigments, a small fraction of the transgenic plants had white flowers, or white sectors on plants. This is not what was expected and is counter-intuitive. Transformation of plants with a gene designed to express more of protein X not only failed to have increased expression of protein X, but in some plants the endogenous gene encoding protein X had also been silenced or supressed. After this phenomenon was discovered with flower color genes, it was shown to be generally applicable with a large number of genes. The general principle behind gene silencing (co-suppression) can be described as follows:
Gene silencing has been used to suppress the expression of ACC synthase in tomato fruits. The strategy is as follows:
This has been developed by a company called DNA Plant Technology (DNAP) in Oakland, California. This company is now owned by a Mexican vegetable seed company called Empressa la Moderna, which controls a large percentage of the vegetable seed market in the US. My understanding is that tomatoes developed from this technology will soon be marketed under the brand name Endless Summer.
I have described various strategies that have been used to develop tomatoes with reduced synthesis of ethylene in fruits. These include metabolism of precursors for ethylene (SAM or ACC), and inactivation of genes for ethylene synthesis by antisense RNA or gene silencing.
How are these going to be used to make fresh market tomatoes with improved flavor and longer shelf life? If these tomatoes make no ethylene, they will not ripen. It is possible to gas these tomatoes with ethylene, but that is little different from current practices and so not a significant advance.
However, if the tomatoes produce only a small fraction of the normal amount of ethylene, then the ripening process will be slowed down. A normal, non-transgenic, mature green fruit might ripen over a period of fifteen days, and be overripe (also called rotten) ten days later. In transgenic tomatoes with reduced ethylene synthesis, this developmental program is extended. Therefore, fruit can be left on the vine longer, allowing them to develop improved flavors and sweetness. When the fruit are harvested, there will still be sufficient time to ship these fruit to market before they rot. In normal fruit, ethylene production is described as being autocatalytic, where exposure to ethylene stimulates production of more ethylene. However, in transgenic fruit these mechanisms to control ethylene synthesis prevent the stimulation of ethylene synthesis by ethylene.
I believe this strategy to control and reduce ethylene production is being used to develop transgenic tomatoes with low ethylene into a successful product. However, I am not aware that any of these are yet on the market in late 1997.