RUI: Vacuolar Transport of Allelochemical Conjugates During Defense Responses.

RUI：防御反应期间化感化学缀合物的液泡运输。

• 批准号: 0114131
• 负责人: John Dean
• 金额: $ 28.88万
• 依托单位: DePaul University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0114131&HistoricalAwards=false
• 关键词: RUI; Vacuolar; Transport; Allelochemical; Conjugates

One of the main functions of the plant cell vacuole is to store allelochemicals. These allelochemicals act as a chemical defense mechanism that helps protect the cell from attacks by other organisms. Allelochemical production presents somewhat of a dilemma for the cell since in order to be effective the plant must produce these compounds in relatively high concentrations. However, since many of these allelochemicals are non-specific toxins, high concentrations in the cell may result in autotoxicity. The plant cells appear to have resolved this dilemma by first conjugating the allelochemical to a water-soluble molecule such as glutathione (GSH) or glucose and sequestering the conjugate in the vacuole. These conjugates may be recognized and transported into the vacuole by a subclass of ATP-binding cassette transporters known as multidrug resistance-associated proteins (MRPs). In order to provide more information regarding the vacuolar transport of allelochemicals, this project has three general objectives. Objective 1 is to determine if glucose or GSH conjugates of trans-cinnamic acid (CA), para-coumaric acid (PC) and salicylic acid (SA) are localized in the vacuole of Arabidopsis cells. This will be determined through in vivo labeling techniques and TLC/HPLC analysis of vacuolar contents. CA, PC and SA were chosen for this study because they are allelochemicals found in virtually all plants. Objective 2 is to develop a simple method for the synthesis of the glucose conjugates of SA, CA and PC. For these compounds, these are the most common conjugates found in plant cells and a facile, high-yield synthesis will provide useful substrates for detailed vacuolar transport studies. Objective 3 is to determine how the vacuolar transport of both glucose and GSH conjugates changes during a defense response. A defense response will be initiated in Arabidopsis cell suspension cultures by the addition of a fungal elicitor or known signal molecules of plant defense (e.g. SA, ethylene, jasmonate, superoxide, and H2O2). Arabidopsis cell suspension cultures will be used for this study because of the molecular tools available and the ease in which the treatments can be added. Changes in the transcription of Arabidopsis MRPs (AtMRPs) in response to the treatments will be followed through dot-blot analysis using the AtMRP expressed sequence tags as probes. Vacuolar transport activity using both glucose and GSH conjugates will be compared to the changes in AtMRP transcription and measured using isolated tonoplast vesicles from the treated cells. These changes in transport activity will also be compared to changes that occur in allelochemical production and conjugation during a defense response. If vacuolar sequestration of allelochemicals is an important aspect of a plant's defense response, then changes in allelochemical production and conjugation should be accompanied by changes in vacuolar transport. The results from these studies should provide considerable insight into the importance of vacuolar sequestration of allelochemicals during a plant cell's defense response. In the future, manipulation of vacuolar sequestration through biotechnology may be able to enhance the plants own natural defense mechanisms, which would have great agricultural significance with regard to pest control.

植物细胞液泡的主要功能之一是储存化感物质。这些化感物质作为一种化学防御机制，有助于保护细胞免受其他生物的攻击。化感物质的产生对细胞来说有点进退两难，因为为了有效，植物必须以相对高的浓度产生这些化合物。然而，由于这些化感物质中的许多是非特异性毒素，细胞中的高浓度可能导致自毒性。植物细胞似乎已经解决了这个难题，首先将化感物质与一种水溶性分子如谷胱甘肽（GSH）或葡萄糖结合，并将结合物隔离在液泡中。这些缀合物可以被称为多药耐药相关蛋白（MRP）的ATP结合盒转运蛋白亚类识别并转运到液泡中。为了提供更多关于化感物质液泡运输的信息，本项目有三个总体目标。目的1是确定葡萄糖或反式肉桂酸（CA）、对香豆酸（PC）和水杨酸（SA）的GSH结合物是否定位于拟南芥细胞的液泡中。这将通过体内标记技术和液泡内容物的TLC/HPLC分析来确定。选择CA、PC和SA进行这项研究，因为它们是几乎所有植物中发现的化感物质。目的二是建立一种简便的合成SA、CA和PC葡萄糖结合物的方法。对于这些化合物，这些是在植物细胞中发现的最常见的缀合物，并且容易、高产率的合成将为详细的液泡运输研究提供有用的底物。目的3是确定在防御反应过程中葡萄糖和GSH结合物的液泡转运如何变化。通过添加真菌激发子或已知的植物防御信号分子（例如SA、乙烯、茉莉酸酯、超氧化物和H2 O2），将在拟南芥细胞悬浮培养物中引发防御反应。拟南芥细胞悬浮培养将用于这项研究，因为分子工具可用，并容易在其中的处理可以添加。将通过使用AtMRP表达的序列标签作为探针的斑点印迹分析来跟踪响应于处理的拟南芥MRP（AtMRP）的转录的变化。将使用葡萄糖和GSH缀合物的质子转运活性与AtMRP转录的变化进行比较，并使用来自处理细胞的分离的液泡膜囊泡进行测量。运输活性的这些变化也将与防御反应期间化感物质产生和结合中发生的变化进行比较。如果化感物质的液泡隔离是植物防御反应的一个重要方面，那么化感物质生产和接合的变化应该伴随着液泡运输的变化。这些研究的结果应该提供相当深入的了解液泡隔离化感物质在植物细胞的防御反应的重要性。未来，通过生物技术操纵液泡隔离可能能够增强植物自身的天然防御机制，这在害虫防治方面具有重大的农业意义。