Robert (Edward Guy Robert) Turgeon - US grants

Leaves of higher plants are converted from sinks to sources of photoassimilate as they mature. This lab has shown that termination of photoassimilate import (end of the sink phase) in tobacco is coincident with, but not due to, achievement of positive carbon balance. This suggests that phloem unloading is symplastic (through plasmodesmata) and ceases when plasmodesmatal connections to surrounding cells are either lost or blocked. Interruption of symplastic flow would also allow the cells of the phloem to achieve the high solute content needed for export by preventing leakage. Prior results support the hypothesis that unloading is passive and symplastic. Additional experiments will be conducted to further substantiate the symplastic phloem unloading route in tobacco sink leaves by testing the effects of breaking plasmodesmatal connections, preventing symplastic transport with high pH and inhibitor treatments, and refining the present morphometric analysis of the unloading pathway to allow stimulation of transport using data on solute flux. High resolution EM studies will be conducted to determine whether plasmodesmata along the unloading pathway become physically obstructed. Phloem loading will also be studied to determine whether an apoplastic step is involved.%%% These studies on the physiology and structure of a developing leaf are providing information on how sugars made during photosynthesis are first stored in leaves and then mobilized to form grains and fruits. This has obvious implications for a better understanding of crop productivity. It is surprising how much plant structure remains to be elucidated. This project is filling an important information gap in plant biology.***//

Four plant growth chambers will be acquired to promote research in the areas of plant physiology, plant molecular biology, and plant biophysics. Specific projects will investigate: 1) developmental physiology in plants; 2) photosynthate partitioning and transmembrane transport; 3) molecular genetics of chloroplast coupling factor; 4) transfer cells and phloem loading; and 5) biophysical aspects of photosynthesis.

9419703 Turgeon Phloem loading is the transport process in which carbohydrate, synthesized by mesophyll cells, is transferred to the vascular tissue (phloem) of leaves in preparation for long-distance translocation to other organs. A new model of phloem loading has been put forward by Dr. Turgeion for those species that translocate stachyose in addition to sucrose. Possibly one-third of all dicotyledonous species are in this group. According to this model, sucrose diffuses from the mesophyll cells into a specialized type of companion cell (the intermediary cell) in the minor veins and is converted there into stachyose. Diffusion occurs through numerous branched plasmodesmata linking bundle sheath cells to intermediary cells. The stachyose accumulates in the intermediary cells and sieve elements because it is too large to diffuse back into the mesophyll. This award supports experimental studies into the dynamics of intermediary cell phlasmodesmata ultrastructure. These plasmodesmata are especially narrow on the intermediary cell side and this constriction is the possible site of discrimination between transport of sucrose in one direction and stachyose in the other. Cryofixation will be used to preserve plasmodesmata structure, which will be studied in mature leaves before and after a reduction in carbohydrate content. Confocal microscope investigations with membrane- specific dyes will determine whether the small vacuoles in intermediary cells, which may be involved in transport of sugar through the cell, originate from the vacuolar system or the endoplasmic reticulum. These studies will concentrate on Cucumis melo and Cucurbita pepo but other stachyose-translocation species may also be employed.

9513727 Harris This is a group travel award to support the participation of U.S. scientists at the 3rd International Workshop on Plasmodesmata, to be held in Zichron Yakov, Israel, March 10 through 15, 1996. ***

This grant provides partial travel support for US participants to Plasmodesma 2001, the fourth international workshop on plasmodesmatal biology, to be held in Cape Town, South Africa, August 19-24, 2001. The workshop will bring together junior and senior scientists from around the world to discuss many aspects of plasmodesmata science, basic and applied. These topics include: plasmodesmata structure and
development, virus movement, solute transport, symplastic domains and regulation, macromolecular trafficking, and gene silencing.
Plasmodesmata research is very active at present and is moving quickly on many fronts. The cross-fertilization of ideas that conferences are noted for is especially important in this multidisciplinary field, where the subject matter and techniques used by one group may be quite foreign to another. A total of
approximately thirty speakers will be invited on the basis of submitted abstracts.
Others will be encouraged to present posters and short talks. The funds from this award will be used to support the travel and per diem expenses of US scientists, most of junior rank (graduate students, postdoctoral fellows, and assistant professors).

This project supports studies on the identification of phloem loading mechanisms in leaves. Sugars manufactured by photosynthesis are loaded into the phloem of minor veins for export to growing organs. There are two known phloem loading mechanisms. Apoplastic loading involves trans-membrane transport while symplastic loading takes place entirely through the plasmodesmata-connected cytoplasm. It is important to be able to distinguish between these mechanisms because they are inhibited by environmental stress, presumably in different ways. According to the current paradigm, apoplastic- and symplastic-loading species can be identified reliably by plasmodesmatal counts. An alternative hypothesis to be tested in this investigation is that apoplastic loading is universal, except for those species that translocate stachyose, or other carbohydrates of similar size, and load by polymer trapping. Phloem loading studies, based primarily on the specific inhibition of trans-membrane transport by p-chloromercuribenzenesulfonic acid, will be conducted on five species that translocate sucrose but have been putatively identified as symplastic loaders on the basis of plasmodesmatal counts. The prediction is that these plants load via the apoplast. Further studies will be conducted to test the hypothesis that plasmodesmatal frequencies in minor vein phloem reflect overall plasmodesmatal numbers in leaves, depending on plant growth habit, and are not necessarily related to phloem physiology.

As efficient collectors of solar energy, plants are subject to damage by light when it is particularly intense, or when harsh environmental conditions prevent the energy from being used productively. Evergreen plants use one of two contrasting strategies to cope with strong light during the winter. Some species protect themselves by completely shutting down photosynthesis and rearranging their light-collecting apparatus to harmlessly dissipate absorbed light. Other species are able to acclimate and remain productive, using solar energy for photosynthesis and growth during brief warm spells. Plants using these two strategies sometimes grow side-by-side in the field. There is speculation that these contrasting responses to environmental change involve differences in the mechanisms by which the products of photosynthesis (sugars) are loaded into the long-distance transport tissue (the phloem) from the leaf cells where they are made. According to this view, species that maintain photosynthetic capacity in winter may be those that use transport proteins for phloem loading, whereas species that downregulate photosynthesis may load via the pores (plasmodesmata) that connect these cell types. Loading via the latter path may be more sensitive to disruption by cold. One representative of each plant type, spinach (with transport proteins) and Mullein (with pores), will be used to study the response to cold temperature under natural conditions in the field and controlled conditions in growth chambers. These species have similar growth forms (rosettes) and retain leaves while overwintering in Colorado. They, as well as additional species of each type (pea, with transport proteins, and pumpkin, with pores) will also be used to study the response of plants when they are transferred from low to high light, which also subjects leaves to potentially damaging, excess energy. An inhibitor of the sugar transport proteins will be used to determine whether species known to export sugars via pores acclimate to cold or high light by altering their sugar export strategy and adding a transport protein component. The focus on these two sugar export strategies will be broadened by characterizing a range of additional structural and ultrastructural features related to carbon export capacity, including the number and cross-sectional area of exporting phloem elements, the density of pore connections, and membrane features associated with the transport proteins. Some or all of these structural features may be augmented to increase carbon export capacity under cold or high light conditions, and it is hypothesized that the downregulating species will exhibit less flexibility in their ability to modulate these structural features. Through these studies a better understanding of sugar export characteristics as potential pivotal determinants of photosynthetic acclimation will begin to emerge. As to the broader impact of this work, this project will (1) involve graduate students in the process of scientific discovery, (2) facilitate a collaboration between disciplines within plant biology and between investigators at two institutions, and (3) allow a better understanding of the underlying mechanisms of the adaptability/resistance of crops and native plants to environmental stress. This will furthermore contribute to a better prediction of changes in plant productivity in response to global change, as well as providing the underlying knowledge necessary for potential increases in the resistance of crops to increased stress through genetic engineering.

The Fifth International Conference on Plasmodesmatal Biology will be held at Asilomar. Topics include plasmodesmata structure and development, macromolecular trafficking, gene silencing, and virus movement. NSF funding will be used to support students and post-doctoral fellows.

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).

Crop yield, responses to high CO2 in the atmosphere, and other aspects of plant growth are partially limited and regulated by the transport of nutrients from leaves to distant plants parts, such as roots and grain. The first step in this process, and an important control point, involves loading sugar and other nutrients into the long-distance transport tissue, the phloem. To date, two phloem-loading mechanisms are known, both of which require metabolic energy. In this project the investigators suggest that there is a third mechanism that does not require energy, one that may be used by as many as half of all flowering plants, especially trees. This could explain why some trees are able to use additional atmospheric CO2 without feedback inhibition, which limits growth of herbaceous crop plants under these conditions. The hypothesis will be tested by genetically engineering poplar trees to inhibit active phloem loading. The prediction is that, although such inhibitory treatments severely retard the growth of plants that load actively, they will have little, if any, effect on poplar. Methods of altering transport through the fine pores that connect plant cells, and serve as conducting channels for virus movement, will also be explored. Broader impacts include, 1) testing and potentially restructuring a key tenet of plant nutrient transport, namely that energy is required to load the phloem in preparation for export; 2) determining how some plants use additional CO2 more effectively than others; and 3) potentially developing a transgenic method of restricting virus movement across specific cellular interfaces. Educational broader impacts include training exceptional undergraduates for careers in science, leading a prison education program, teaching a large introductory biology course for non-biology majors, and writing scientific articles for the general public.

The phloem transports nutrients, developmental signals and defense compounds throughout the plant body. It is often assumed that the phloem is a homogeneous tissue. However, on the basis of preliminary data the investigators suggest that, in the small veins of leaves where transported materials are loaded into the phloem, the network is heterogeneous in its structure and biochemistry, with specific functions assigned to distinct subsets of phloem cells. This hypothesis will be tested in several species by a number of techniques. Both light and electron microscopy will be used to quantify cell-specific features involved in transport, including the channels (plasmodesmata) that connect the phloem to surrounding cells. Cell-specific promoters (the DNA elements that control gene expression) will be used to identify cell types within veins and this information will be used in conjunction with microscopy to profile gene expression patterns in the different cell types. In a third project, specific phloem cells will be selectively killed with a toxin synthesized under the control of cell-specific promoters and these plants will be used to obtain and characterize phloem sap to identify nutrients, macromolecules and defense compounds transported in individual cell types. In terms of broader impacts, specific cells involved in the synthesis and transport of compounds required for the proper development and health of crop plants will be identified. Educational broader impacts include the incorporation of experimental hypotheses and results in undergraduate course material, employment of undergraduate students in phloem sap studies, and continued participation in the Cornell Prison Education Program. RNA sequence data will be deposited at the National Center for Biotechnology Information and processed data files will be uploaded to iPlant.