Tonoplast transport as a determinant of tomato fruit chemical composition

The aim of this proposal is to investigate factors controlling the chemical composition of tomato fruit, a crop of major economic importance worldwide. Both the flavour and nutritional quality of tomatoes are determined by the chemicals that accumulate during fruit ripening, yet we have only a limited understanding of how this process is controlled. In mature fruit, the cells are dominated by a compartment called the central vacuole, which contains most of the sap in fleshy fruit. This compartment can occupy as much as 95 % of the cell's volume, the remaining 5 % being taken up by the cell cytoplasm and outlying cell wall. As the tomato fruit grows, chemicals such as sugars, organic acids and amino acids are produced in the cytoplasm. They are then removed from their site of synthesis by transport into the central vacuole across the bounding membrane surrounding this compartment, called the tonoplast. But this traffic is not all one-way. As the fruit ripens, some solutes leave the vacuole to be re-metabolized in the cytoplasm, with other solutes moving back into the vacuole to compensate. Thus, the composition of the mature fruit is a complex outcome of metabolic events in the cytoplasm combined with transport of solutes across the tonoplast membrane. Whereas the pathways of basic metabolism in fruit cells are well understood, we have much less knowledge of the transport proteins that reside in the tonoplast membrane. In fact, we have indirect evidence that these proteins may play a much more important role in determining fruit composition than previously suspected. As the first part of this project, therefore, we shall isolate the tonoplast membrane from tomato fruit at defined stages during their development and analyse its protein content by mass spectrometry. This will provide a valuable inventory of proteins residing in the tonoplast membrane, and of their changes in abundance during the ripening process. By correlating these changes with the chemical composition of the fruit, we should obtain the first clues as to which tonoplast proteins are important in regulating transport across the vacuolar membrane. In another strand of the project, we will use a genetic approach to obtain independent information on factors controlling fruit composition. A powerful resource for this purpose is provided by the natural genetic variation found between cultivated tomatoes and their close relatives in the wild. Indeed, several of these species are sufficiently closely related that they can be hybridized. By analysing the characteristics of the progeny of such crosses (e.g. with respect to fruit composition), it is possible to make deductions about which genes may be contributing to particular traits. Using this approach, we will investigate whether any of the genes correlated with differences in fruit composition encode likely tonoplast membrane proteins. If they do, we will cross-reference this list against the information on tonoplast proteins obtained by mass spectrometry. This will allow us to focus on a limited number of the most promising candidates for more detailed characterization. In the final part of the project, we will test the function of the selected candidate proteins directly to determine, first, whether they indeed reside in the tonoplast membrane in intact cells, and second, what solutes they are capable of transporting into and out of the vacuole. We will focus on candidate transporters of organic acids and amino acids, as these are important determinants of fruit flavour and acidity that have been little investigated to date. The combination of the protein identification and genetic approaches promises to yield important new information on the factors determining fruit composition. This will also be valuable for directing future breeding strategies towards the selection of new elite lines with improved fruit traits, without the need for intervention using genetic modification techniques.

The aim of this work is to identify mechanisms controlling the accumulation of primary metabolites in tomato fruit, with a focus on six major carboxylic acids and amino acids that influence fruit quality. We have obtained evidence that the control of fruit composition resides less in the capacity of central metabolism than previously supposed. Rather, we hypothesize that transport proteins at the tonoplast membrane play an important role in regulating solute accumulation, as most of the major metabolites are ultimately sequestered in the large central vacuole of developing fruit cells. Additionally, vacuolar contents are dynamic during fruit development, with certain solutes effluxing from the vacuole and others accumulating as the fruit matures. However, little is known about these transporters and the way they are regulated in a cellular context. We shall first undertake a quantitative proteomics study and investigations of transport activity on tonoplast membrane isolated at five defined stages of tomato fruit development. This will provide an inventory of candidate transporter proteins and will reveal whether their abundance correlates with changing fruit metabolite content. In parallel, we will use a genetic approach to identify candidate transporters by analysing two sets of introgression lines between cultivated tomato and wild relatives. Candidate transporters that show allelic diversity between the parent species and map to regions associated with differences in fruit metabolite content will be cross-referenced to the tonoplast proteome. A number of transporters will then be selected for detailed functional characterization, (a) to determine their subcellular localization, and (b) to investigate their transport properties in appropriate expression systems. Finally, the most promising metabolite transporters will be tested by overexpression in transgenic plants to determine if they produce the predicted changes in chemical composition of the fruit.