Last time I described a number of approaches to develop tomatoes with reduced synthesis of ethylene, as a method to alter the ripening of tomatoes so they will have improved quality as fresh market tomatoes. A second aspect of tomato fruit ripening that is a target for modification is the softening of the fruit. There are two reasons why this is of interest.
First, it is possible that fruit that soften more slowly could be harvested after they have started to ripen on the vine, but still shipped and marketed before they have started to deteriorate. This would allow the fruit to develop more flavor on the vine, an important trait that would improve consumer acceptance.
Second, altering the softening of tomato fruit might alter the processing properties of the fruit to improve the efficiency and/or quality of the final processed product.
As I described in the last lecture, ethylene is responsible for inducing this cascade of genes that are involved in fruit ripening. This includes genes involved in softening of the fruit. This raises an important question: What is the biochemistry of fruit softening? As with all the other processes that occur during fruit ripening, softening of the fruit is a highly controlled developmental process, it is not just a process of deterioration or decay.
The first question to ask about fruit softening is what makes the fruit firm in the first place. Firmness of the fruit is a function of the properties of cell walls, the structural component that surrounds every plant cell. Cell walls are comprised of:
- cellulose fibres
- pectins
- hemicelluloses
- proteins
I am not an expert in cell wall structure (far from it), but models of how these various components are assembled in cell walls have been developed. In this model, the cellulose fibres are aligned parallel to one another and are linked together by the other components. These crosslinks between the cellulose fibres act in a manner similar to crossmembers in a bridge. Without these crossmembers, the bridge would collapse very easily. Addition of the crossmembers makes the bridge relatively rigid. If you assemble a square from 2 by 4's, it is very easy to push it out of shape. Add a diagonal crossmember and you make this a stronger, more stable structure. The crosslinking components of the cell walls (pectins, hemicelluloses and proteins) make the cellulose fibres more rigid.
This model of cell wall structure must also be able to account for the normal processes that occur during plant growth and development, including cell expansion and division, and other phenomena such as leaf abscission and fruit softening.
During fruit ripening, various enzymes that degrade specific components of the cell wall are synthesized in the fruit. Among the enzymes that accumulate in the fruit are cellulases (to break down cellulose), and polygalacturonase (PG) and pectin methylesterase (PME), both of which are involved in breakdown of the pectin crosslinking molecules. These enzymes contribute to the softening of the fruit by reducing the rigidity of the cell wall structures. As mentioned above, expression of the genes encoding these enzymes is regulated by ethylene.
Calgene, one of the first agricultural biotechnology companies based in Davis, California, as well as others proposed that reducing the expression of polygalacturonase (PG) in tomato fruit would slow down the softening of the fruit. They used the antisense RNA approach to produce plants with reduced expression of PG in fruit. You should now be able to lay out the strategy to do this:
It was claimed that softening of the fruit was slowed in these fruit, allowing them to remain on the vine longer, with harvest later than the typical "mature green fruit" stage. The tomatoes could then be shipped and marketed before they turned to mush.
This was the basis for the FLAVR SAVR tomato, the first whole food product of biotechnology. After several reviews by regulatory agencies, this product was launched in late 1994. FLAVR SAVR tomatoes were available under the brand name "Macgregor tomatoes" for a few months. However, the product was not a commercial success and was withdrawn from the market after less than one year.
There are several reasons why this highly touted product of biotechnology failed, and I don't know the answer for sure. One possibility is that the product did not perform as claimed in terms of slowing the softening of the fruit. I have been told by knowledgeable sources that others who have used antisense RNA to reduce the expression of PG in ripening fruit have been unable to produce fruit that either ripen more slowly or remain any firmer than normal fruit. Why would Calgene have continued with the development of this product if it was not performing as desired? I don't know the answer to this question either. Perhaps the transgenic plants they produced had lower levels of PG than others had been able to obtain, leading to delayed softening. Or maybe their reasons to continue with this product had less to do with the technology and more to do with the economics of a small biotechnology company that needed to attract investors. The company had very little income because most of its products were still in the development phase. Admitting that their first product in this area had to be abandoned might have led some investors to withdraw their support. I stress this is all speculation on my part, but it may help to explain why the FLAVR SAVR tomato failed after such a long development period.
An alternative explanation is that Calgene was unable to put in place the high quality growing, packing, and shipping infrastrucure to handle this material that required more delicate treatment than standard tomatoes.
In the end, Calgene was acquired by Monsanto. I don't believe that Monsanto was interested in the FLAVR SAVR business. Instead, Calgene had also been involved in a number of other areas of plant biotechnology, including development of Roundup resistant plants by expression of a bacterial gene, and the development of Brassica oilseed crops with altered oil compositions which have special applications in the food industry and elsewhere. Monsanto was able to acquire these and at the same time head off possible patent litigation from Calgene over how to make plants resistant to Roundup.
Reducing expression of PG did not have the expected effect on fruit softening, and perhaps this reflects our current limited understanding of the biochemistry of the cell wall and how this changes during fruit ripening. Nevertheless, others have tried essentially the same antisense RNA approach to produce plants with low expression of pectin methylesterase (PME) in the fruit, again using antisense RNA. As its name implies, PME is involved in metabolism of pectins in the cell wall. Pectins in mature green fruit are long polymers, and PME is expressed during fruit ripening to break these large polymers into shorter molecules. It is likely that PME is one of the first enzymes involved in the metabolism of pectins.
Transgenic plants with reduced PME levels ripen normally. This is on reflection not surprising, as nothing has been done to interfere with ethylene production, the central regulator of fruit ripening. However, the pectins in these fruit with reduced PME activity remain large as the fruit ripen. They are not broken down into shorter pectins because there is no PME activity. As a result of this change in metabolism during ripening, the pectins remain as large polymers.
The outcome of this modification is that using this more viscous juice as the starting material means less processing is required to produce tomato paste of the desired consistency. This has great potential for reducing the cost of processing these tomatoes, and perhaps improving the quality of their final product. Avtar Handa in the Purdue Horticulture department is one of the leading researchers in this area. Further research is in progress to see if tomato fruit with reduced expression of PME can be developed into a successful product. And, while the fruit with reduced expression of PG were not successful in improving the quality of fresh tomatoes, reducing the expression of PG in fruit does increase the soluble solids content of tomatoes, likely by increasing the size of pectins. Tomato paste processed from these fruit is on the market in Britain.
Another approach to manipulating plant responses to ethylene, including fruit ripening and flower senescence, is based on blocking the perception of the hormone. To understand how this might function, we must first consider how ethylene is able to bring about these changes in plants. The role of ethylene can be described simply as follows:
This diagram could be modified to account for many hormone responses. However, it does not explain how ethylene produces these chages in gene expression. Does ethylene move to the nucleus and directly alter the binding of proteins that regulate gene expression? No, there are very few examples of such direct interactions between hormones and the transcription process. Instead, a series of biochemical steps are involved in moving this signal from outside the cell to the nucleus. This is diagrammed below:
This is known as a signal transduction pathway, able to transduce the signal, in this case ethylene, from the outside the cell to a response brought about by changes in gene expression. (Please note that not all of these changes are always mediated by altering gene expression.)
The ethylene receptor has now been cloned from a number of plants. The ethylene receptor is a protein that sits in the membrane and has a site for binding ethylene on the outside of the cell. When ethylene binds to the receptor, the signalling pathway is activated leading to changes in gene expression. Arabidopsis mutants have been found that are unable to respond to ethylene, and one class of these ethylene-insensitive mutants have a defective ethylene receptor that is unable to transmit the signal to the nucleus when ethylene is present. Somewhat surprisingly, this is a dominant mutation. Even when functional ethylene receptors are present in the cell, as in a heterozygote, they are unable to function. In some way, the normal ethylene receptors have been "poisoned" by the mutant receptor.
This has led to another strategy to modify ethylene responses in transgenic plants, where the signal transduction pathway is blocked by expressing this dominant mutant ethylene receptor.
Transgenic plants (tomato and petunia) have been produced that express a dominant mutant ethylene receptor, either from Arabidopsis or tomato. Some of these transgenic plants have been shown to be insensitive to ethylene. As a result various ethylene responses are blocked:
While fruit that are incapable of ripening are of little use, this does demonstrate that the action of ethylene can be blocked in transgenic plants. However, there is a lot of commercial interest in developing flowers with delayed senescence, both for the cut flower market and for ornamental plants. Development of this approach into a commercial product has not yet occured, but I think it likely that we will see flowers with delayed senescence as a result of one or more of these methods to manipulate ethylene metabolism or perception in transgenic plants.
In summary, many important processes in plants are regulated by ethylene, especially those involved with post-harvest processes.
Why have so many different methods been developed to alter responses to ethylene? One reason is that once a specific method has been patented, it may not be possible for someone else to use that method. The patent holder may be unwilling to license the technology to another party, or they may want too much money to allow use of this method. Patent protection may be a stimulator of invention in this area. In addition, there may be some methods that work better in specific circumstances, to modify particular traits.
A final comment on manipulating ethyene: I have focused most of my discussion of how to manipulate ethylene responses on the tomato, in part because it is such an important vegetable crop and has been the focus of a great deal of attention. However, there are many other crops where ethylene responses are of critical importance. Examples include other fruits where ripening is controlled by ethylene, such as melons, bananas, peaches, raspberries and many more. And as I mentioned earlier, flower senescence, which determines the vase life of cut flowers, can also be changed by manipulating ethylene responses. So, while most of the attention has focused so far on tomatoes, I think it very likely this will change in the next few years as this technology is applied to a wider range of crops.