Tonoplast transport as a determinant of tomato fruit chemical composition

这项提议的目的是研究控制番茄果实化学成分的因素，番茄果实是世界上重要的经济作物。西红柿的风味和营养品质都是由果实成熟过程中积累的化学物质决定的，但我们对这一过程是如何控制的了解有限。在成熟的果实中，细胞主要由一个叫做中央液泡的隔室控制，它包含了肉质果实中的大部分汁液。这个区室可以占据细胞体积的95%，剩下的5%被细胞质和外围细胞壁占据。随着番茄果实的生长，细胞质中会产生糖类、有机酸和氨基酸等化学物质。然后将它们从它们的合成位点转移到中央液泡中，穿过包围着这个腔室的边界膜，称为张力质体。但这种交通并不都是单向的。随着果实的成熟，一些溶质离开液泡在细胞质中重新代谢，另一些溶质返回液泡进行补偿。因此，成熟果实的组成是细胞质代谢事件和溶质在细胞质膜上运输的复杂结果。尽管水果细胞的基本代谢途径已经被很好地理解，但我们对存在于细胞质膜上的转运蛋白知之甚少。事实上，我们有间接证据表明，这些蛋白质在决定水果成分方面的作用可能比之前猜测的要重要得多。因此，作为该项目的第一部分，我们将在番茄果实发育的特定阶段分离出番茄质体膜，并用质谱法分析其蛋白质含量。这将提供一个有价值的蛋白质库存驻留在细胞质膜，并在成熟过程中其丰度的变化。通过将这些变化与果实的化学成分联系起来，我们应该获得关于哪些叶绿体蛋白在调节跨液泡膜运输中起重要作用的第一个线索。在该项目的另一个方面，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。在栽培番茄和其野生近亲之间发现的自然遗传变异为这一目的提供了有力的资源。事实上，这些物种中有几个是近亲，它们可以杂交。通过分析这些杂交后代的特征（例如，关于果实组成），就有可能推断出哪些基因可能对特定性状起作用。利用这种方法，我们将研究是否有任何与果实成分差异相关的基因编码可能的质体膜蛋白。如果有，我们将与质谱法获得的细胞质蛋白信息进行交叉比对。这将使我们能够专注于有限数量的最有希望的候选者进行更详细的表征。在项目的最后一部分，我们将直接测试所选候选蛋白的功能，首先，确定它们是否确实存在于完整细胞的细胞质膜中，其次，确定它们能够将哪些溶质运输进出液泡。我们将重点关注有机酸和氨基酸的候选转运蛋白，因为这些是迄今为止很少研究的水果风味和酸度的重要决定因素。蛋白质鉴定和遗传方法的结合有望对决定果实成分的因素产生重要的新信息。这也将有助于指导未来的育种策略，选择具有改良果实性状的新优良品系，而无需使用转基因技术进行干预。

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.