EAGER: Defining the phloem-mobile proteins of Arabidopsis

在高等动物中，血液通过静脉和动脉在全身流动，携带营养物质、激素和信号分子，协调不同器官的功能。植物有一种类似的液体，称为韧皮部汁液，它通过韧皮部导管将糖从光合作用器官运送到植物的其他部位，并在遥远的植物器官之间传递有关营养需求、压力或疾病的信息。科学家们对韧皮部汁液介导的信号传导只有一个初步的了解，主要是因为收集不被周围细胞的液体严重污染的韧皮部汁液样本是极其困难的。虽然有证据表明，韧皮部汁液中存在数百种蛋白质，但只有一小部分蛋白质被证实确实可以运动，而且只有一种蛋白质具有已知的功能。在这个项目中，研究人员将识别数百种韧皮部汁液蛋白质，这些蛋白质证实存在长距离移动。研究人员的新方法包括将两种具有不同遗传序列的植物嫁接在一起，然后使用蛋白质序列分析来识别植物1在植物2的韧皮部导管中的蛋白质。如果成功，该项目将产生第一个可移动的韧皮部汁液蛋白质的大型数据集，克服目前植物内部信号传递领域的瓶颈。这些蛋白质的同一性将催生新的研究方向，并将影响大量的植物生物学家。韧皮部信号是植物在生物和非生物胁迫下生存能力的重要组成部分。尽管韧皮部移动蛋白在植物内部的通讯具有令人兴奋的潜力，但目前对于大多数植物中韧皮部汁液蛋白质组的识别还没有达成共识，包括模式植物拟南芥，在那里研究人员可以最好地测试这些蛋白质的功能。PI将使用一种嫁接方法来确定通过移植物连接进行长距离移动的蛋白质。利用1000多份拟南芥种质的广泛序列数据，PI将识别序列变异导致~1100个预测的韧皮部汁液蛋白氨基酸差异的嫁接伙伴。将进行异种嫁接，将从接穗中收集富含韧皮部汁液的蛋白质样本，并将使用质谱仪来鉴定已进入接穗组织的砧木蛋白质。该项目产生的蛋白质组将依赖于观察到的蛋白质运动。这与该领域以前基于直接提取韧皮部汁液来定义韧皮部汁液蛋白质组的尝试形成了鲜明对比，这一过程极其困难，而且存在技术困难。数百种可移动蛋白质的定义将克服这一领域目前的瓶颈，现有的韧皮部汁液蛋白质组是相互矛盾的，并且公认具有大样本人工制品。一旦掌握，韧皮部汁液蛋白质组将导致基于可移动蛋白的身份和功能的关于韧皮部汁液信号转导的可检验的假说。

In higher animals, blood moves throughout the body in veins and arteries, carrying nutrients, hormones and signaling molecules that coordinate the functions of different organs. Plants have an analogous fluid called phloem sap, which moves through phloem vessels to carry sugars from photosynthetic organs to the rest of the plant, and to communicate information about nutritional needs, stress or disease between distant plant organs. Scientists have only a rudimentary understanding of phloem sap-mediated signaling, primarily because it is extremely difficult to collect a phloem sap sample that is not heavily contaminated by fluids from the surrounding cells. While evidence suggests that hundreds of proteins exist in the phloem sap, only a small number are confirmed to actually move and only one has a known function. In this project, the researchers will identify hundreds of phloem sap proteins with confirmed long distance movement. The researcher's novel approach involves grafting two plants with distinct genetic sequences together and then using protein sequence analysis to identify proteins from plant 1 in the phloem vessels of plant 2. If successful, this project will generate the first large dataset of mobile phloem sap proteins, overcoming the current bottleneck in the field of intra-plant signaling. The identity of these proteins will catalyze new research directions and will impact a large number of plant biologists. Phloem signaling is an essential component of the plant's ability to survive biotic and abiotic stresses. Therefore, this project will suggest new strategies for improving crop plant growth and yield.Despite the exciting potential of intra-plant communication via phloem-mobile proteins, there is no current consensus as to the identity of the phloem sap proteome in most plants, including the model plant Arabidopsis thaliana where researchers could best test the function of these proteins. The PI will use a grafting approach to definitively identify proteins that move long distance through a graft union. Taking advantage of the extensive sequence data available for over 1000 Arabidopsis accessions, the PI will identify grafting partners where sequence variation leads to amino acid differences in ~1100 predicted phloem sap proteins. Heterografts will be made, protein samples enriched for phloem sap will be collected from the scion, and Mass Spectrometry will be used to identify rootstock proteins that have travelled to the scion tissue. The proteome generated by this project will rely on observed protein movement. This is in contrast to previous attempts in the field to define the phloem sap proteome based on direct phloem sap extraction, a process that is extremely difficult and plagued with technical difficulties. The definition of hundreds of mobile proteins will overcome the current bottleneck in this field, where the existing phloem sap proteomes are contradictory and generally acknowledged to have large sampling artefacts. Once in hand, the phloem sap proteome will lead to testable hypotheses about phloem sap signaling based on the identity and function of the mobile proteins.