Tonoplast transport as a determinant of tomato fruit chemical composition

该提案的目的是调查控制番茄果实化学成分的因素，番茄果实是全球重要的经济作物。番茄的风味和营养质量都是由果实成熟过程中积累的化学物质决定的，但我们对这一过程的控制了解有限。在成熟的果实中，细胞由称为中央液泡的隔室控制，其中包含肉质果实中的大部分汁液。这个区室可以占据多达95%的细胞体积，剩余的5%被细胞质和外围细胞壁占据。随着番茄果实的生长，细胞质中会产生糖、有机酸和氨基酸等化学物质。然后，它们通过运输进入中央液泡而从其合成位点被移除，穿过围绕该隔室的边界膜，称为液泡膜。但这种交通并不都是单向的。当果实成熟时，一些溶质离开液泡在细胞质中重新代谢，而另一些溶质则回到液泡中进行补偿。因此，成熟果实的组成是细胞质中的代谢事件与溶质穿过液泡膜的运输相结合的复杂结果。虽然在果实细胞中的基本代谢途径是很好的理解，我们有少得多的知识，在液泡膜上的运输蛋白。事实上，我们有间接的证据表明，这些蛋白质可能在决定水果成分方面发挥着比以前怀疑的更重要的作用。因此，作为本项目的第一部分，我们将在番茄果实发育过程中的特定阶段从番茄果实中分离出液泡膜，并通过质谱法分析其蛋白质含量。这将提供一个有价值的清单的蛋白质驻留在液泡膜，其丰富的变化，在成熟过程中。通过将这些变化与果实的化学组成相关联，我们应该获得关于哪些液泡膜蛋白在调节跨液泡膜的运输中是重要的第一线索。在该项目的另一条链中，我们将使用遗传方法来获得控制水果成分的因素的独立信息。一个强大的资源，为这一目的提供了自然的遗传变异之间发现的栽培番茄和它们的近亲在野外。事实上，这些物种中有几个是足够密切相关的，它们可以杂交。通过分析这种杂交后代的特征（例如果实组成），可以推断哪些基因可能对特定性状有贡献。使用这种方法，我们将调查是否有任何基因与水果成分的差异编码可能的液泡膜蛋白。如果他们这样做，我们将交叉引用这个名单对信息的液泡膜蛋白质获得质谱。这将使我们能够专注于数量有限的最有希望的候选人进行更详细的表征。在该项目的最后一部分，我们将直接测试所选候选蛋白质的功能，以确定，首先，它们是否确实存在于完整细胞的液泡膜中，其次，它们能够运输哪些溶质进出液泡。我们将集中在候选运输有机酸和氨基酸，因为这些都是水果风味和酸度的重要决定因素，迄今为止很少调查。蛋白质鉴定和遗传方法的结合有望产生重要的新信息的因素决定水果的组成。这也将是有价值的指导未来的育种策略，选择新的精英线与改善水果性状，而不需要使用遗传修饰技术的干预。

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.