Genes and proteins for solute transport and sensing.

Transport processes are required for nutrient acquisition, translocation in the plant and for compartmentation within the cells. The weed Arabidopsis occurs in many environmentally distinct locations around the world, and a significant proportion of the genome of this small plant encodes membrane proteins, especially transport proteins and putative sensors that cope with these conditions. Many of these proteins are homologous to transporters from other organisms including bacteria, fungi and animals. This chapter provides an overview of the components and mechanisms responsible for solute transport and sensing. Described are the basic principles of transport processes and the transporters of the pump and carrier classes found in Arabidopsis. Transport of the most prominent solutes that are taken up and distributed within the plant such as nitrogenous compounds (ammonium, nitrate, amino acids, peptides) and carbohydrates such as sugars (sucrose, glucose) or sugar alcohols (e.g. mannitol) are discussed in some detail. General characteristics of transport processes Cellular plasma membranes demark the interface between life and death: they protect the highly structured and organized plant cellular interior, the cytosol, from the hostile external environment. This creates a compartment which permits biochemical reactions to be carried out within a protected domain. Such a barrier, however, aside from its protective role, must at the same time allow passage of nutrients and solutes to allow cellular functions to proceed. Acquisition of ions, transfer of metabolites and excretion of waste products, but also transport of chemoattractants, kayromones (chemicals that convey information about interactions), hormones and substances that help mobilizing nutrients (e.g. protons, organic acids and phytosiderophores), establish homeostasis between the interior and the exterior. Furthermore, the plasma membrane represents the interface to the environment and thus must play a crucial role in relaying information about the external environment. Depending on the position of a cell in the plant, this may either be the soil, or the cell wall space as the interface to adjacent or distant cells of the same or a different organism (e.g. pathogenic or symbiotic bacteria and fungi). The plasma membrane itself is composed of membrane lipids and integral and peripheral proteins (for a detailed discussion see Gennis, 1989). Protein composition within the plasma membrane can vary in wide ranges between 20% and 80% (dry weight). Furthermore membranes may contain carbohydrates in the form of glycolipids or glycoproteins. In the aqueous environment of living cells, the hydrophobic hydrogen-carbon tails of membrane lipids avoid water contact and, although lacking attracting forces, self-assemble and cluster together into an energy minimized state. The polar headgroups orient to the surrounding water on both sides, whereas the hydrophobic tails orient towards each other on the inside leading to the formation of a bilayer consisting of two leaflets with a thickness of ∼4nm (Fig. 1). Biological membranes are characterized by an asymmetric distribution of lipids between the two leaflets, i.e. restriction of phosphatidylserine to the inner leaflet (Boon & Smith, 2002; Manno et al., 2002). The lipid asymmetry may have a variety of important biological functions and a loss of asymmetry can be used as a measure for cell death (Schlegel and Williamson, 2001). Figure 1. Model of a lipid bilayer containing both embedded transmembrane (glyco-)proteins and peripheral (glyco-)proteins associated with the membrane or anchored to it. Schematic drawing based on the Singer-Nicolson fluid mosaic model. Besides the plasma membrane, the eukaryotic cell also contains intracellular compartments surrounded by internal membranes, which enclose specialized organelles and vesicles, separating functional units within a single cell. Such lipid bilayers form a diffusion barrier preventing free exchange of molecules between the in- and outside of the cellular compartments. The lipid composition of the various membranes in a plant cell can vary significantly leading to differences in the membranes properties. Furthermore the phospholipid, glycolipid and sterol composition of cellular membranes is acclimated in different environments, especially due to altered temperature in order to maintain fluidity and functionality. The lipid bilayer forms a lipid “sea”, in which molecules can normally move freely in lateral direction. In other words, individual lipid molecules and peripheral, integral and lipid anchored proteins may diffuse and distribute randomly, whereas an asymmetrical distribution of lipids on the in- and outside of the membrane is generated and maintained by active processes (“flip-flop”, for details confer Gennis, 1989). The accepted model for a biological membrane is that of a fluid mosaic (Fig. 1). The free diffusion of some membrane proteins, however, is restricted by cytoskeletal anchors or association with other membrane proteins, and the resulting patchy localization of proteins leads to polarized cell structures. In fungi, free diffusion of lipids and membrane proteins may be restricted by diffusion barriers, generated by septins, whereas in animals tight junctions serve to compartmentalize the plasma membrane. So far, such diffusion barriers in plants have not been identified at the molecular level, e.g. no septin homologs were found in the Arabidopsis genome, but it would be very surprising if plants would not contain such barriers. Although little is known about details of specific lipid-protein interactions, membrane lipids may be involved in regulation (Robl et al., 2001) and targeting (Bagnat et al., 2001) of plasma membrane proteins. Due to the existence of specialized intercellular connections in plant cells, the plasmodesmata, the plasma membrane of most cells within a plant seems to be continuous between adjacent cells. Exceptions are specialized cells such as guard cells, which lose their intercellular connections during development, and cells or organs that are not connected by plasmodesmata, like pollen or seed. Since membrane proteins, however, are often restricted to one of two adjacent cells, barriers similar to those found in fungal or animal cells must exist that prevent free diffusion of membrane proteins between cells. All molecules have an inherent capacity to passively traverse membranes, but permeability coefficients vary over several orders of magnitude. Usually this permeability is correlated with the solubility of the substance within the hydrophobic environment of the membrane bilayer (Gennis, 1989). Thus non-charged and hydrophobic molecules permeate more easily than ions and polar substances across a membrane. However, in most cases, passive movement is too limited to be relevant for biological processes. Thus transport proteins facilitate the movement of specific molecules across membranes, achieving rates from few molecules per second up to about 108s−1, the latter value approximating the diffusional limit in aqueous solution. Although small molecules such as water and urea display relative high permeability coefficients, their transport across biological membranes is nevertheless facilitated by transport proteins, which is also the case for hydrophobic substances like lipids or cholesterol. The composition of a given membrane is not constant, but rather the transport properties of cellular membranes are continuously adjusted to control and optimize metabolite exchange according to the cellular needs. This is achieved by acclimating the types of transporters present at the membrane to the requirements by biogenesis of new carriers via transcriptional up-regulation on the one hand and removal of unsuitable carriers on the other hand (e.g. turnover, Kuhn et al., 1997). Furthermore transport activity can be regulated very rapidly by modulating their activity by posttranslational protein modifications, by withdrawal due to endocytosis or by the release from internal compartments.