A New Approach to the Study of Symplastic Phloem Loading

将营养物质从叶子运送到果实和块茎等可收获器官的第一步是将它们装载到长距离运输组织韧皮部。这是一个代谢活跃的过程，在韧皮部产生非常高浓度的营养物质，主要是糖。水通过渗透作用，由于细胞壁是刚性的，压力上升。这种压力是汽车轮胎的十倍以上，它驱动着长距离的水流，就像房子里的水压驱动着花园水管里的水一样。因此，负荷是养分运动的动力，是农业产量的直接决定因素。第二，已知韧皮部负荷的物种特异性机制。一种是外体负荷，蔗糖离开光合作用的叶细胞，进入细胞壁空间（外体）。然后通过跨膜转运蛋白将能量泵入韧皮部细胞。在第二种加载机制中，蔗糖从其起始点扩散到韧皮部，通过连接它们的狭窄孔（胞间连丝）从一个细胞传递到另一个细胞。以扩散为基础的过程似乎不太可能将糖浓缩到细胞中。然而，这个项目的假设是第二个系统是通过“聚合物捕获”来运作的。根据这一假说，韧皮部中的蔗糖分子被转化为更大的糖，如棉子糖和水苏糖，由于它们的大小，它们无法通过胞间连丝扩散回去。糖因此变得浓缩，韧皮部压力上升。许多生长旺盛的植物，比如南瓜，就使用这个系统。聚合物陷阱模型得到了几条证据的支持，包括某些物种韧皮部中异常高数量的胞间连丝与棉子糖和水苏糖的运输之间的严格关联。然而，进一步的实验受到了限制，因为这些物种中没有一个能轻易地用外源DNA进行转化。最近发现，棉子糖/水苏糖植物Verbascum phoeniceum可以很容易地通过标准技术进行转化。因此，测试聚合物陷阱模型成为可能。首先，对植物进行基因工程改造，使其在外质体中产生酵母转化酶。转化酶降解蔗糖。在外质体装载体中，这种处理严重干扰装载，因为转运蛋白对蔗糖是特异性的。然而，有一种预测认为，由于蔗糖从未进入外质体，因此在V. phoeniceum中负荷不会受到损害。在第二组实验中，通过rna干扰技术将蔗糖转运体基因下调。同样，这种处理消除了载脂蛋白的负荷，但预计对V. phoeniceum的影响很小，因为转运蛋白不参与。在第三组实验中，棉花糖和水苏糖的合成将在V. phoeniceum中下调。这里的预期是，负荷将被严重抑制，因为植物不能制造出将碳水化合物困在韧皮部所需的糖，也没有办法通过渗透产生压力。更广泛的影响：PI相当重视大众媒体的科学写作。PI的一名研究生，Sarah Davidson，已经在传统的植物生物学领域开始了一个非传统的科学传播项目，并与传播系合作。其目的是在研究生院开辟一条永久的新路，让其他学生追随。PI实验室最近发表的其他文章包括麦克米伦参考百科全书“生物学”中关于韧皮部运输的条目，以及生物教师广泛阅读的《生物科学》杂志上关于韧皮部装载的评论文章（正在准备中）。

The first step in transporting nutrients from leaves to harvestable organs, such as fruits and tubers, is to load them into the long-distance transport tissue, the phloem. This is a metabolically active process that creates within the phloem a very high concentration of nutrient, primarily sugar. Water follows by osmosis, and since the cell walls are rigid, pressure rises. The pressure, more than ten times that of an automobile tire, drives long-distance flow, just as the water pressure in a house drives water along a garden hose. Thus, loading is the motivating force for movement of nutrients, and is a direct determinate of agricultural yield. Two, species-specific mechanisms of phloem loading are known. In one, apoplastic loading, sucrose exits photosynthetic leaf cells and enters the cell wall space (apoplast). It is then energetically pumped into phloem cells by transmembrane transporter proteins. In the second loading mechanism, sucrose diffuses from its point of origin into the phloem, passing from cell to cell through narrow pores (plasmodesmata) that join them. It may seem unlikely that a process based on diffusion could concentrate sugar in cells. However, the hypothesis of this project is that this second system operates by "polymer trapping." According to this hypothesis, sucrose molecules in the phloem are converted to larger sugars, raffinose and stachyose, that are unable to diffuse back through the plasmodesmata because of their size. Sugar thus becomes concentrated, and the phloem pressure rises. Many vigorously growing plants, such as pumpkins, use this system.The polymer trap model is supported by several lines of evidence, including a strict correlation between extraordinarily high numbers of plasmodesmata in the phloem of certain species, and the transport of raffinose and stachyose. However, further experimentation has been limited by the fact that none of these species has been known to be readily transformable with foreign DNA. It was recently discovered that Verbascum phoeniceum, a raffinose/stachyose plant, can be easily transformed by standard techniques. Hence, it has become possible to test the polymer trap model.First, plants will be genetically engineered to produce yeast invertase in the apoplast. Invertase degrades sucrose. In apoplastic loaders, this treatment severely interferes with loading since the transporter proteins are specific for sucrose. However, a prediction is that loading will not be compromised in V. phoeniceum since sucrose never enters the apoplast. In the second set of experiments, sucrose transporter genes will be down regulated by RNA-interference technology. Again, this treatment abolishes loading in apoplastic loaders but is predicted to have little affect in V. phoeniceum, since transporters are not involved. In the third set of experiments, synthesis of raffinose and stachyose will be down regulated in V. phoeniceum. The expectation here is that loading will be severely inhibited because the plant cannot make the sugars needed to trap carbohydrate in the phloem and there will be no way to generate pressure by osmosis.Broader Impacts: The PI places considerable emphasis on science writing for the mass media. One of the PI's graduate students, Sarah Davidson, has begun a non-traditional, program of science communication within the traditional Field of Plant Biology and in collaboration with the Communication Department. The intention is to pave a permanent, new track in graduate school that other students will follow. Other recent articles from the PI's lab include an entry on phloem transport for MacMillan's reference encyclopedia "Biology" and a review article on phloem loading for BioScience, a journal widely read by biology teachers (in preparation).