Progress 08/15/05 to 08/14/10
Outputs
OUTPUTS: The primary objective of this proposal was to determine how sugar alcohols are loaded into the phloem. However, as the work proceeded, we expanded the goals to include a comprehensive picture of phloem loading strategies in all plants, including the two major types of carbohydrates loaded: sucrose and sugar alcohols. We speculated that the mechanism of sugar alcohol loading varies in different species. In plants with few plasmodesmatal connections between the photosynthetic cells and phloem we predicted that sugar alcohols would load through the apoplast by specific transporters. In species with abundant plasmodesmata, we speculated that loading would be symplastic. The results supported our predictions. The results demonstrate that Plantago major loads sucrose and sorbitol via the apoplast. This is active loading using and it increases the concentration of both compounds in the phloem. Apple (Malus domestica) loads both sucrose and sorbitol symplastically. It is a passive process. Asarina scandens uses a combination of mechanisms. It loads most of its sucrose apoplastically, and mannitol symplastically. We then undertook a comprehensive analysis of 45 species. Our results demonstrate that there are three loading strategies, apoplastic, polymer trapping and passive. The passive strategy is symplastic with no concentrating step. The strategies can be distinguished on the basis of solute concentrations, plasmodesmatal abundance and uptake of radiolabeled solute into the minor veins. The results indicate that passive loading is more prevalent that previously recognized, especially in trees. This study provides a comprehensive picture of phloem loading mechanisms and suggests that loading strategies can be determined routinely in species of interest. In a third project, we cloned a maltose transporter gene from apple that mediates maltose transfer across the chloroplast inner envelope membrane. We studied its expression profile, its localization to chloroplasts and the complementation of Arabidopsis lines deficient in this transporter activity. In further work in the last (extended) year of the grant we initiated a project on poplar. There is a great deal of current interest in poplar as a biofuel species. It is our hypothesis, based on our previous studies, that poplar phloem loads passively and does not use sucrose transporters directly in this process. To test this hypothesis we have been generating transgenic poplar plants in which the sucrose transporters are downregulated by RNAi. At the time of this writing we have over 100 transgenics in hand but they are not quite large enough to conduct the physiological tests. We expect that the physiology will be complete within six months. We also published a review in the Annu Rev of Plant Biology, and an opinion piece in Plant Physiology. PARTICIPANTS: Robert Turgeon (PI): experimental design, training. Edwin Reidel (Post-doctoral Fellow): studies on apple and Plantago Emilie Rennie (undergraduate): survey of 45 species, including carbohydrate analysis and 14C-distribution, including Asarina. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Phloem loading is the first step in translocation and the one that creates the hydrostatic pressure needed to drive mass flow through the sieve tubes from source to sink organs. We believe that this is a major regulatory step as well, at least for certain loading strategies. Therefore, to understand how carbon partitioning is controlled, and to devise rational strategies for increasing biomass production, phloem-loading mechanisms must be understood in more than a handful of model species. We believe that the work funded by this grant provides a way to easily establish loading mechanisms in any species, including trees. It will also provide a framework for understanding the role that loading plays in the control of carbon partitioning, growth rate, responses to environmental perturbations and certain types of pollution, and to elevated carbon dioxide.
Publications
- No publications reported this period
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: The primary objective of this proposal was to determine how sugar alcohols are loaded into the phloem of leaves in preparation for long distance transport to sink tissues, such as roots and fruits. However, as the work proceeded, we expanded the goals to include a comprehensive picture of phloem loading strategies in all plants, including the two major types of carbohydrates loaded: sucrose and sugar alcohols. Based on comparative anatomy we speculated that the mechanism of sugar alcohol loading varies in different species. In plants with few plasmodesmatal connections between the photosynthetic cells and phloem, we predicted that sugar alcohols would load through the apoplast by specific transporters. In species with abundant plasmodesmata, we speculated that loading would be symplastic. We tested these hypotheses in three species and the results supported our predictions. The results demonstrate that Plantago major loads sucrose and sorbitol via the apoplast. This is active loading using proton-symporters and it substantially increases the concentration of both compounds in the phloem. Apple (Malus domestica) loads both sucrose and sorbitol symplastically. It is a passive process driven by high concentrations of both compounds in the mesophyll cells of the leaf. Asarina scandens uses a combination of mechanisms. It loads most of its sucrose apoplastically, and mannitol symplastically. Some sucrose is loaded symplastically by the polymer trapping mechanism discovered in this lab. We then undertook a more comprehensive analysis of over 45 species, assessing loading strategies for sucrose, raffinose-family oligosaccharides, and sugar alcohols. Our results demonstrate that loading there are three loading strategies, apoplastic, polymer trapping and passive. The passive strategy is symplastic with no concentrating step. The strategies can be distinguished on the basis of leaf solute concentrations, plasmodesmatal abundance and the capacity of leaf discs to take up radiolabeled solute into the minor veins. Uptake of radiolabel into minor veins is analyzed by autoradiography. The results indicate that passive loading is more prevalent that previously recognized, especially in trees. This study provides a comprehensive picture of phloem loading mechanisms and suggests that loading strategies can be determined routinely in species of interest. In a third project, we cloned a maltose transporter gene from apple that mediates maltose transfer across the chloroplast inner envelope membrane. We studied its expression profile, its localization to chloroplasts and the complementation of Arabidopsis lines deficient in this transporter activity. PARTICIPANTS: Robert Turgeon (Professor and PI): responsible for experimental design, training. Lailiang Cheng (Professor): responsible for experimental design, training. Edwin Reidel (Post-doctoral Fellow): physiological studies on apple and Plantago. Dr. Reidel came to us trained primarily in physiology and horticulture. He received molecular biology training while conducting this research. He is now employed at Neuman Seed Co. in Oregon. Emilie Rennie (undergraduate): survey of 45 species, including carbohydrate analysis and 14C-distribution, including Asarina. Ms. Rennie was trained in radiolabeling techniques, HPLC and autoradiography as well as plant growth. She is currently in the PhD program in Plant Biology at the Univ. of California at Berkeley. Veronique Amiard (Post-doctoral Fellow): Electron microscopy. Dr Amiard learned physiological techniques as well as electron microscopy. She is currently a research scientist in the Plant Biotechnology unit, Agro Aquaculture Nutritional Genomic Center, National Research Agriculture Institute, CRI-Carillanca, Temuco, Chile. TARGET AUDIENCES: The major target audience for this research is the international community of agricultural scientists, especially those interested in crop productivity. The PI has also made a significant effort to reach out to broad and diverse audiences to disseminate results and teach fundamental aspects of biology. He helped found the Cornell Prison Education Program and is a member of the board of directors of the program. He teaches a one-semester course in Introductory Biology each year at the Auburn Correctional Facility, a maximum security prison near Ithaca, NY. The inmates in this program receive credit toward an Associates degree conferred by Cayuga Community College. The PI has also taught Introductory Biology to Cornell non-majors and majors for 29 years. During the last grant period he wrote a major review for plant biologists PROJECT MODIFICATIONS: None.
Impacts
Phloem loading is the first step in translocation and the one that creates the hydrostatic pressure needed to drive mass flow through the sieve tubes from source to sink organs. We believe that this is a major regulatory step as well, at least for certain loading strategies. Therefore, to understand how carbon partitioning is controlled, and to devise rational strategies for increasing biomass production, phloem-loading mechanisms must be understood in more than a handful of model species. We believe that the work funded by this grant provides a way to easily establish loading mechanisms in any species, including trees. It will also provide a framework for understanding the role that loading plays in the control of carbon partitioning, growth rate, responses to environmental perturbations and certain types of pollution, and to elevated carbon dioxide.
Publications
- Turgeon, R., Wolf, S. (2009) Phloem transport: cellular pathways and molecular trafficking. Annu. Rev. Plant Biol. 60: 207-221.
- Reidel, E. J., Turgeon, R., and Cheng, L. (2008). A maltose transporter from apple is expressed in source and sink tissues and complements the Arabidopsis maltose export-defective mutant. Plant Cell Physiology 49: 1607-1613.
- Reidel, E.J., Rennie, E.A., Amiard, V., Cheng, L., Turgeon, R. (2009) Phloem loading strategies in three plant species that transport sugar alcohols. Plant Physiol. 149: 1601-1608.
- Rennie, E.A., Turgeon, R. (2009) A comprehensive picture of phloem loading strategies. Proc. Natl. Acad. Sci. (USA) 106: 14162-14167.
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: The primary objective of this proposal was to determine how sugar alcohols are loaded into the phloem. However, as the work proceeded, we expanded the goals. We are now working toward a comprehensive picture of phloem loading strategies in all plants, including the two major types of carbohydrates loaded: sucrose and sugar alcohols. All the experimental work is complete and we are now writing the papers. The first paper, based on comparative anatomy, loading of 14C-labeled compounds into veins, and quantitative carbohydrate analysis, will deal with sugar alcohols and sucrose in three representative species, apple, Plantago and Asarina. Apple loads both sucrose and sorbitol symplastically. It is a passive process driven by high concentrations of both compounds in the mesophyll cells of the leaf. Plantago loads sucrose and sorbitol via the apoplast. This is active loading using symporters and it substantially increases the concentration of both compounds in the phloem. Asarina uses a combination of mechanisms. It loads most of its sucrose apoplastically, and mannitol symplastically. Some sucrose is loaded symplastically by the polymer trapping mechanism discovered in this lab. The patterns of loading fit nicely with the anatomy of the minor vein phloem in these species. For example, there are large numbers of plasmodesmata in apple and in one type of minor vein phloem in Asarina. Plasmodesmata are sparse in Plantago and in the second type of minor vein phloem in Asarina. The second paper is a comprehensive analysis of over 40 species. Starting from thermodynamic principles, we derived a theoretical, two-dimensional matrix that describes all possible combinations of loading strategies. The results of this survey fit the model without exception; all species sort into the matrix cells as predicted. Distinct phenotypic correlations are evident that could not otherwise be assumed. For example, no species with high sugar alcohol levels exhibit vein loading in autoradiographs. The results provide a comprehensive picture of phloem loading mechanisms and suggest that loading strategies can be determined routinely in species of interest. In a third project, we cloned a maltose transporter gene from apple that mediates maltose transfer across the chloroplast inner envelope membrane. We studies its expression profile, its localization to chloroplasts and the complementation of Arabidopsis lines deficient in this transporter activity. PARTICIPANTS: Robert Turgeon (PI): experimental design, training. Edwin Reidel (Post-doctoral Fellow): studies on apple and Plantago. Training in microscopy, analytical chemistry, molecular biology, radiolabeling and transport physiology. Emilie Rennie (undergraduate): survey of over 40 species, including carbohydrate analysis and 14C-distribution. Training in transport physiology, analytical chemistry, 14C-labeling procedures. Emilie is currently a PhD student in the Plant and Microbial Biology Department at UC Berkeley. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Phloem loading is the first step in translocation and the one that creates the hydrostatic pressure needed to drive mass flow through the sieve tubes from source to sink organs. We believe that this is a major regulatory step as well, at least for certain loading strategies. Therefore, to understand how carbon partitioning is controlled, and to devise rational strategies for increasing biomass production, phloem-loading mechanisms must be understood in more than a handful of model species. We believe that the work funded by this grant provides a way to easily establish loading mechanisms in any species, including trees. It will also provide a framework for understanding the role that loading plays in the control of carbon partitioning, growth rate, responses to environmental perturbations and certain types of pollution, and to elevated carbon dioxide.
Publications
- Reidel, E. J., Turgeon, R., and Cheng, L. (2008). A maltose transporter from apple is expressed in source and sink tissues and complements the Arabidopsis maltose export defective mutant. Plant Cell Physiology (in press).
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Progress 08/15/06 to 08/14/07
Outputs
The overall objective of this proposal is to determine how sugar alcohols are loaded into the phloem. We have completed one major project toward this objective - an analysis of sorbitol and sucrose loading in the phloem of apple leaves - and we are preparing the paper for submission. Although current thinking on sugar alcohol loading leans heavily toward an apoplastic route, we find that the characteristics of loading in apple leaves favor a symplastic pathway. In other words, we find no evidence for active phloem loading in this species; both sucrose and sorbitol simply diffuse from mesophyll cells to the minor vein phloem. A second project is almost complete and will probably be ready for submission early this fall. We suggest that all phloem loading strategies for sucrose and sugar alcohols, and combinations of these compounds in species that translocate both, can be determined on the basis of three simple assays. Starting from thermodynamic principles, we derived a
theoretical, two-dimensional matrix that describes all possible combinations of loading strategies. We have conducted experiments on over 40 dicotyledonous plants. On the basis of these results, all species sorted into the matrix cells as predicted. Distinct phenotypic correlations were evident that could not otherwise be assumed. For example, no species with high sugar alcohol levels exhibit vein loading in autoradiographs. The results provide a comprehensive picture of phloem loading mechanisms and suggest that loading strategies can be determined routinely in species of interest. In a third project we have obtained transgenic tobacco and Verbascum phoeniceum plants expressing the ononitol synthase gene and we are using these plants to study alternate pathways of sugar alcohol loading in species that use apoplastic and symplastic pathways.
Impacts
The loading of sucrose and raffinose-family oligosaccharides is now reasonably understood. Sugar alcohols are the last remaining piece in the phloem-loading puzzle. When we determine how these compounds enter the phloem, it should be possible to put together a comprehensive picture of photoassimilate loading in all its forms. Sugar alcohols themselves constitute the major carbohydrate transported in apples and the other woody Rosaceae, as well as several other important crop plants. Manipulation of sink-source relations in these plants, a key to improving crop yield, requires a fundamental understanding of all the transport steps between the chloroplast and sink cells, and this will only be possible once we know how sugar alcohols enter the phloem.
Publications
- No publications reported this period
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Progress 08/15/05 to 08/14/06
Outputs
We described three objectives in the Experimental Design: 1) to produce and study the transport properties of transgenic tobacco and Verbascum plants that synthesize ononitol in either the mesophyll or companion cells, 2) to determine whether sugar alcohols accumulate against a thermodynamic gradient in jasmine, Euonymus and celery, and 3) to clone mannitol transporters from jasmine. At this early stage in the grant cycle, considerable progress has been made in made on the first objective. The constructs are complete and transgenic plants have been obtained from culture. We are currently screening these plants for expression levels. Within the next month the plants will be large enough to begin transport experiments. The second objective has taken a somewhat different direction. In preliminary tests we have found that we can predict which plants will accumulate radiolabeled sugars or sugar alcohols in the minor vein phloem based on simple measurements of osmolality,
sugar concentration, and sugar profiles. For example, species that rely on diffusion to load the phloem always have high sugar, or sugar alcohol levels. Species that use the polymer trap mechanism synthesize raffinose and stachyose. Apoplastic loaders translocate sucrose, and in some cases sugar alcohol, but have overall low levels of these compounds in the lamina, reflective of low levels in the mesophyll. We are conducting a survey of 42 species of dicots to test the validity of these generalizations. Work on the third objective has not yet begun.
Impacts
The loading of sucrose and raffinose-family oligosaccharides is now reasonably understood. Sugar alcohols are the last remaining piece in the phloem-loading puzzle. When we determine how these compounds enter the phloem, it should be possible to put together a comprehensive picture of photoassimilate loading in all its forms. Sugar alcohols themselves constitute the major carbohydrate transported in apples and the other woody Rosaceae, as well as several other important crop plants. Manipulation of sink-source relations in these plants, a key to improving crop yield, requires a fundamental understanding of all the transport steps between the chloroplast and sink cells, and this will only be possible once we know how sugar alcohols enter the phloem.
Publications
- No publications reported this period
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