A New Approach to the Study of Symplastic Phloem Loading

Cornell University, Ithaca NY

Investigators

Abstract

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).