A New Approach to the Study of Symplastic Phloem Loading

将营养从树叶运输到水果和块茎等可收获器官的第一步是将它们装载到长途运输组织-韧皮部。这是一个代谢活跃的过程，在韧皮部内产生了非常高浓度的营养物质，主要是糖。水伴随着渗透作用，由于细胞壁是坚硬的，压力就会上升。这种压力是汽车轮胎的十倍以上，可以驱动长距离流动，就像房子里的水压沿着花园的软管驱动水一样。因此，负荷是养分移动的动力，是农业产量的直接决定因素。第二，物种特有的韧皮部加载机制是已知的。其一，质外体加载，蔗糖离开光合作用的叶细胞，进入细胞壁空间(质外体)。然后，它被跨膜转运蛋白高能泵入韧皮部细胞。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气孔(胞间连丝)从一个细胞传递到另一个细胞。基于扩散的过程似乎不太可能在细胞中浓缩糖。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，棉子糖和水苏糖，由于它们的大小，这些糖不能通过胞间连丝扩散回来。糖因此变得集中，韧皮部压力上升。许多生长旺盛的植物，如南瓜，都使用这种系统。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极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，进一步的实验受到这样一个事实的限制，即这些物种中没有一个是可以很容易地与外来DNA转化的。最近发现，棉籽糖/水苏糖植物Verbascum pheniceum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已成为可能。首先，将对植物进行基因工程，使其在质外体中产生酵母转化酶。转移酶能降解蔗糖。在质外体加载器中，这种处理严重干扰了加载器的加载，因为转运蛋白是蔗糖所特有的。然而，一种预测是，由于蔗糖永远不会进入质外体，因此在凤毛委陵菜中装载不会受到影响。在第二组实验中，蔗糖转运蛋白基因将通过RNA干扰技术下调。同样，这种处理取消了质外体加载器的加载，但预计对凤冠花的影响很小，因为不涉及转运蛋白。在第三组实验中，棉子糖和水苏糖的合成将在凤凰山弧菌中下调。这里的预期是，负载将被严重抑制，因为植物无法制造将碳水化合物捕获到韧皮部中所需的糖，也将无法通过渗透作用产生压力。更广泛的影响：PI相当重视为大众媒体撰写科学文章。PI的一名研究生Sarah Davidson已经开始了一个非传统的、在传统植物生物学领域内与传播部合作的科学交流项目。这样做的目的是为研究生院铺平一条永久的、新的轨道，让其他学生也能效仿。PI实验室最近的其他文章包括麦克米兰的参考百科全书《生物学》中关于韧皮部运输的条目，以及一篇关于生物科学的韧皮部装载的评论文章，这是一本生物教师广泛阅读的期刊(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).