Andreas Weber

Research

Plastids are the metabolic factories of plant cells. They are the site of an enormous variety of biosynthetic functions including the synthesis of starch, isoprenoids, phenolic compounds, fatty acids and the assimilation of carbon dioxide, sulfate and ammonia into organic compounds.

The great variety of metabolic functions carried out by plastids requires an enormous traffic of metabolites across the plastid envelope membrane.

My lab is studying the transport processes that connect the metabolic pathways in plastid and cytosol, in particular those transporters involved in carbon- and nitrogen-metabolism, using biochemistry, molecular biology, and molecular plant physiology.

Transport of Dicarboxylic Acids

Plastids are the main sites of ammonia assimilation in plant cells. Ammonia is resulting from a variety of metabolic reactions in plant cells and its assimilation into organic compounds is of outmost importance for plant growth and development:

Figure 1. Sources of ammonia in plant metabolism

The assimilation of ammonia in plastids is dependent on an organic precursor molecule - 2-oxoglutarate. We have identified transporters in the chloroplast envelope membrane that catalyze the uptake of 2-oxoglutarate into the plastid (2-oxoglutarate/malate translocator, DiT1) and the export of glutamate from the plastid (glutamate/malate translocator, DiT2).

Figure 2. Simplified scheme of the biosynthetic pathway of 2-oxoglutarate and its transport in to plastids by DiT1.

Transport of Carbohydrates

Plants store a significant part of recently assimilated carbon dioxide transiently as transitory starch in the chloroplast stroma. Transient starch is degraded during the dark period to provide plant metabolism with carbohydrates in the absence of photosynthesis. In many plant species, the degradation of starch occurs through the hydrolytic pathway, yielding mainly glucose and maltose as end products. These carbohydrates have to be exported from the plastid stroma to the cytosol. We have identified a candidate gene for the plastidic glucose transporter (pGlcT) and we are currently in the progress of identifying the maltose transporter (pMalT).

Figure 3. A simplified representation of hydrolytic starch breakdown and the export of breakdown products from plastids by a glucose (GlcT) and a maltose transporter (MalT).

Functional Studies of Membrane Transporters

It is very difficult to purify membrane transporters from plastid envelope membranes in a functional state. Therefore, we express the corresponding cDNAs in heterologous expression hosts such as bakers yeast and purify the recombinant proteins from the yeast cells. To study the kinetic constants and other biochemical properties of the transport proteins, we reconstitute them into lipid vesicles, so called proteo-liposomes. The liposome system can be considered equivalent to a photometric test of enzyme activities.

The use of heterologous expression systems allows the production of large amounts of highly purified membrane proteins. In the future, we want to use our expression system to produce recombinant membrane proteins for structural studies.

Knockout Mutants and Transgenic Plants with Decreased Transporter Activities

In addition to our studies of the biochemical properties of metabolite transporters in vitro, we would like to know more about there function in living plants (in vivo). To achieve that goal, we are screening for T-DNA insertion mutants in Arabidopsis thaliana, and we are generating transgenic plants with reduced activity of metabolite transporters using antisense and RNAi technology. Transgenic and knockout plants are analyzed at the multiple levels such as their metabolite contents, enzymatic activities, transcript levels, and physiological parameters (photosynthesis, gas exchange, biomass production etc.).

Regulation of Metabolic Networks in Plant Cells

The complex metabolic networks in plant cells are distributed over several sub-cellular compartments such as cytosol, ER, Golgi, plastids, vacuole, mitochondria, and peroxisomes. Metabolite transporters in the membranes surrounding the compartments interconnect the pathways in the sub-cellular compartments. It is a very interesting question whether the flux of metabolites between sub-cellular metabolic pathways is limited or controlled by the activity of metabolite transporters. To address this question, we analyze transgenic plants and mutant plants with reduced or increased activities of metabolite transporters. Using the concept of control analysis, we will be able to quantify the control coefficients that metabolite transporters have on a particular metabolic pathway.

Metabolic Engineering of Plant Metabolism

MSU is a center of excellence for metabolic engineering of plant metabolism (http://www.plantmetabolism.msu.edu/). In comparison to microbial cells, metabolic engineering of plant metabolism is particularly challenging because of the high degree of compartmentation of plant cells. Metabolite transporters that are located in the membranes surrounding the organelles connect metabolic pathways in the sub-cellular compartments of a plant cell. Using genetic methods, we can engineer sub-cellular metabolite pools by increasing or decreasing the activity of metabolite transporters in the organellar membranes. Thereby, we can re-direct metabolic fluxes in plant cells, a major step towards a rational design of metabolic pathways in plant cells.

Before we can do effective biotechnological engineering of crop plants to increase their commercial value and their nutritional benefits, we need to understand the underlying biology of metabolic pathways and their regulation. Our research seeks to enhance our knowledge base of transport processes in plant cells. The knowledge gained through our research will contribute to the long-range goal of a sustainable agriculture by providing the basic knowledge that is required for rational engineering of plant metabolism.

Genome Analysis of Galdieria sulphuraria

Extreme environments are frequently inhabited by extremophiles, which are almost always prokaryotic organisms. However, the red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs (pH 0.05 to 4; temperatures up to 56 degrees C). The gene sequences of this living fossil should reveal much about the evolution of modern eukaryotes. Galdieria thrives on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols - evidence of a large repertoire of metabolic genes equaled by few other organisms and a potentially rich source of thermo-stable enzymes for biotechnology. Moreover, this organism tolerates toxic metal ions such as cadmium, mercury, aluminum or nickel, suggesting potential use in bioremediation. Proteins isolated from thermophiles frequently crystallize more readily, making Galdieria proteins valuable for structural biology studies. Finally, the high temperatures under which Galdieria photosynthesizes are at the extreme ranges for this process; thus this organism will likely prove a useful model for physical studies on the photosynthetic apparatus. The genome of Galdieria sulphuraria will be sequenced. Sequences will be collated and annotated, provided to public databases, and specialized databases for the exploitation of this data will be developed. This research will generate a wealth of knowledge on the adaptation of eukaryotic organisms to extreme environments, on molecular mechanisms of protein thermo-stability, and on the coordination of gene regulation in adaptation of metabolic pathways to extreme stress. In addition, the project will add to the understanding of genome evolution, and the phylogeny of plants and plastids.

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Selected Recent Publications

Voll, L.M., Allaire, E.E., Fiene, G., Weber, A.P.M. (2004) The Arabidopsis thaliana phenylalanine insensitive growth (pig) mutant exhibits a deregulated amino acid metabolism. Plant Physiol. 136: 3058-3069.

Weber, A.P.M., Oesterhelt, C., Gross, W., Bräutigam, A., Imboden, L.A., Krassovskaya, I., Linka, N., Truchina, J., Schneidereit, J., Voll, L.M., Zimmermann, M., Riekhof, W.R., Yu, B., Garavito, M.R., Benning, C. (2004) EST-analysis of the thermo-acidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts. Plant Mol. Biol., 55: 17-32.

Weber, A.P.M. (2004) Solute transporters as connecting elements between cytosol and plastid stroma. Curr. Opin. Plant Biol., 7: 247-253.

Weber, A.P.M., Schneidereit, J. & Voll, L.M. (2004) Using mutants to probe the in vivo function of plastid envelope membrane metabolite transporters. J. Exp. Bot. 55: 1231-1244.

Sharkey, T.D., Laporte, M., Lu, Y., Weise, S.E. & Weber, A.P.M. (2004) Engineering Plants for Elevated CO₂: A Relationship between Sugar Sensing and Starch Degradation. Plant Biol., 6 (3): 280-289.

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Current Funding

National Science Foundation (NSF) Collaborative Proposal "Genome Analysis of Galdieria sulphuraria -a unique thermo-acidophilic photosynthetic microorganism". (2003-2005; Michael Garavito and Christoph Benning co-PIs).

National Science Foundation: Collaborative Proposal "Role of Plastidic Dicarboxylate Translocators in Plant Ammonia Assimilation". (2004-2007; Marianne Huebner and Vince Melfi co-PIs).

US Department of Energy: Collaborative Proposal "Maltose Biochemistry and Transport in Leaves". (2004-2007; Thomas D. Sharkey co-PI).

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Teaching

Andreas Weber teaches the following course:

PLB 301, Introductory Plant Physiology.

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Lab Members