Genes and Proteins for Solute Transport and Sensing

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The Arabidopsis Book 2002 American Society of Plant Biologists

Genes and proteins for solute transport and sensing

Uwe Ludewig and Wolf B. Frommer

ZMBP, Plant Physiology, Universitt Tbingen, Tbingen, Germany

email: [email protected], [email protected]

http://www.uni-tuebingen.de/plantphys

INTRODUCTION

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 cellularfunctions 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 environ-

ment. 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).

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

The Arabidopsis Book 2002 American Society of Plant Biologists

First published on September 30, 2002 doi: 10.1199/tab.0092: e0092.


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Figure 1. Model of a lipid bilayer containing both embedded transmembrane (glyco-)proteins and peripheral (glyco-)proteins associated withthe membrane or anchored to it. Schematic drawing based on the Singer-Nicolson fluid mosaic model.

on the in- and outside of the membrane is generated andmaintained by active processes (flip-flop, for detailsconfer Gennis, 1989). The accepted model for a biologicalmembrane is that of a fluid mosaic (Fig. 1). The freediffusion of some membrane proteins, however, isrestricted by cytoskeletal anchors or association with

other membrane proteins, and the resulting patchylocalization of proteins leads to polarized cell structures.In fungi, free diffusion of lipids and membrane proteinsmay be restricted by diffusion barriers, generated byseptins, whereas in animals tight junctions serve tocompartmentalize the plasma membrane. So far, suchdiffusion barriers in plants have not been identified at themolecular level, e.g. no septin homologs were found in the

Arabidopsis genome, but it would be very surprising ifplants would not contain such barriers. Although little isknown 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 plasmamembrane proteins.

Due to the existence of specialized intercellular

connections in plant cells, the plasmodesmata, theplasma membrane of most cells within a plant seems tobe continuous between adjacent cells. Exceptions arespecialized cells such as guard cells, which lose theirintercellular connections during development, and cells ororgans that are not connected by plasmodesmata, likepollen or seed. Since membrane proteins, however, are

often restricted to one of two adjacent cells, barrierssimilar to those found in fungal or animal cells must existthat prevent free diffusion of membrane proteins betweencells.All molecules have an inherent capacity to passively

traverse membranes, but permeability coefficients vary

over several orders of magnitude. Usually thispermeability is correlated with the solubility of thesubstance within the hydrophobic environment of themembrane bilayer (Gennis, 1989). Thus non-charged andhydrophobic molecules permeate more easily than ionsand polar substances across a membrane. However, inmost cases, passive movement is too limited to berelevant for biological processes. Thus transport proteinsfacilitate the movement of specific molecules acrossmembranes, achieving rates from few molecules persecond up to about 108s-1, the latter value approximatingthe diffusional limit in aqueous solution. Although smallmolecules such as water and urea display relative highpermeability coefficients, their transport across biologicalmembranes is nevertheless facilitated by transport

proteins, which is also the case for hydrophobicsubstances like lipids or cholesterol.

The composition of a given membrane is not constant,but rather the transport properties of cellular membranesare continuously adjusted to control and optimizemetabolite exchange according to the cellular needs. Thisis achieved by acclimating the types of transporters


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Genes and Proteins for solute transport and sensing 3 of 32

present at the membrane to the requirements bybiogenesis of new carriers via transcriptional up-regulation on the one hand and removal of unsuitablecarriers on the other hand (e.g. turnover, Khn et al.,1997). Furthermore transport activity can be regulatedvery rapidly by modulating their activity byposttranslational protein modifications, by withdrawal dueto endocytosis or by the release from internalcompartments.

Structure of membrane proteins

Due to the difficult biochemistry of hydrophobicmembrane proteins, only little is known about the exacttree-dimensional molecular structure of most membraneproteins, but integral membrane proteins are thought tocontain mostly either -barrel or -helical domains withinmembrane environment (Ikeda et al., 2002; Mller et al.,

2001). Solute transport proteins embedded within the lipidbilayer must have defined properties: they must form (inmost cases) a partially hydrophilic pore, through whichsolutes can move in a controlled manner, but must betightly sealed against the membrane lipids to preventpassage of solutes along this interface. The fewcrystallized and resolved structures are mostly frombacterial origin, but strong homology suggests that thestructures apply also for the eukaryotic relatives. Knownstructures include potassium (Doyle et al., 1998), chloride(Dutzler et al., 2002) and water channels (Murata et al.,2000; Sui et al., 2001), P-type pumps (Toyoshima et al.,2000), Na+/H+ antiporters (Williams et al., 1999), and ABC-transporters (Chang & Roth, 2001; Locher et al., 2002)mitochondrial proteins involved in respiration or redox

reactions and the bacterial rhodopsin (Davies et al., 2001).Furthermore lower resolution data are available for anumber of transporters such as EmrE, NhaA, OxlT, TetA,Band3, (for review cf. Veenhoff et al., 2002). As a generalfeature, major parts of these proteins are formed by -helical domains, connected via short extramembraneousloops. A surprising diversity of functions has evolved bysuch an ordered alignment of amino acids and secondarystructures. -barreled structures are also observed andoccur mainly in proteins from the outer bacterialmembrane such as maltoporin or iron siderophoretransporters (Ferguson et al., 2002; Cowan et al., 1992)but also in the outer mitochondrial membrane. Since theaverage thickness of the membrane is ~4 nm, thedomains which span the membrane must have a length of

at least 4 nm to cross the membrane. If they are tiltedrelative to the perpendicular angle of the surface, theymust even be longer. In order to span the membranecompletely, an -helix must consist of 18-22 amino acidresidues. The amino acids in such an -helix arepreferentially hydrophobic to be able to interact with thehydrophobic lipid environment in order to seal the

interface tightly. However, to the inside of the pore, theymust be hydrophilic to allow passage of hydrophilicsolutes. Thus most of the helices in a membrane spanningsegment must be amphipathic, i.e. hydrophilic on theinside and hydrophobic to the outside. To be able to forma selective pore in the membrane, several helices arebundled together and packed tightly. Such structures canbe generated either by combining multiple polypeptideswith single transmembrane spanning domains, or (as inmost transporters) several such domains can be fusedinto a single polypeptide forming a so-called polytopicmembrane protein. Most of the transporters inArabidopsis are thought to belong to the polytopic -helixclass. This highly modular structure of amino acidcomposition in membrane proteins allowed thedevelopment of algorithms that predict whether a givenprimary gene sequence encodes for a polytopicmembrane protein. Furthermore this allowed tosystematically predict the presence and composition ofmembrane proteins in various genomes (e.g. bacteria,yeast, Arabidopsis). The simplest way to identify aputative transporter in the genome is to search forstretches of hydrophobic amino acids. Moresophisticated, in some cases self-learning programs havebeen developed that enable a relative high confidencelevel prediction (Sonnhammer et al., 1998). Web-basedprediction programs include TmHMM_v2.0 (Krogh et al.,2001), HmmTop_v2.0 (Tusnady and Simon, 1998, TMap(Persson and Argos, 1996), TopPred_v2.0 (Claros and vonHeijne, 1994), TMPred (Hofmann and Stoffel, 1993),SosuiG_v1.1 (Mitaku and Hirokawa, 1999) andEiconda_v0.9 (Schwacke, unpublished).

In the case of most solute transporters, it is oftennecessary to rely on such predictions, since so far nothree-dimensional structures of metabolite transporters

such as sugar or amino acid transporters are available.Based on the outputs of prediction programs andcomparison with available structural data, one mayhypothesize that membrane transporters in eukaryotestypically consist of several -helical domains, while -barrels predominate in the structures of porins in the outermembrane of mitochondria, reflecting their prokaryoticorigin.

Using the seven prediction programs listed above, anestimate of total integral membrane proteins in

Arabidopsis can be given. Considering only proteins withone or two transmembrane regions (predicted by at least6 of the 7 programs), proteins with three or moretransmembrane regions (predicted by at least 5 of the 7programs) and excluding proteins with a single predicted

N-terminal transmembrane region with a cleavable signalpetide, 6475 proteins (25 %) of the 25492 predictednuclear encoded proteins can be classified astransmembrane (TM) proteins (Schwacke et al.,submitted; Arabidopsis Genome Initiative, 2000). Typicalmembrane transporters have between 6-14 hydrophobicspans that may cross the membrane. About 5% of all


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Figure 2. From sequence to structure. A: Amino acid sequence of AtSUT2 (At2g02860). Stretches of hydrophobic amino acid stretches thatare predicted to be embedded into the membrane are shown in bold. B. Prediction of transmembrane topology for the sucrose transporterAtSUT2 by TmHMM v2.0. C. Schematic two-dimensional view of AtSUT2. D. Predicted 3-dimensional structure of a distant AtSUT2homolog, the human hexose transporter GLUT1, which belongs to the MFS-family (major facilitator superfamily) (Zuniga et al., 2001). The 12-helical transmembrane helices are predicted to tilt slightly. E. Space filling representation of the main (in yellow) and auxiliary (in blue)substrate pore of GLUT1, reference as before. With kind permission from The American Society for Biochemistry and Molecular Biology. F.The functional unit of sucrose transporters may be a dimer.


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genes in Arabidopsis are predicted to have at least sixtransmembrane domains. The most typical structure asoriginally identified in the prototype of all solutetransporters, lac permease (Kaback et al., 2001) consistsof 12 transmembrane helices, a topology that is found forplant sugar transporters as well (Sauer 1989; Riesmeier etal., 1992; for review cf. Veenhoff et al., 2002). Interestinglyfragments of such proteins can be expressed together toreconstitute functional transporter activity (Zen et al.,1994, Wrubel et al., 1990), Reinders et al., 2002a, Xie etal., 2000), demonstrating that the packaging of the helicesis mainly due to forces between the individual helices (fora detailed discussion cf. Veenhoff et al., 2002).

Membrane proteins are integrated in a definedorientation into the membrane. Prediction programs takeinto account differences in the amino acid distribution inhelices with respect to the two sides of the membrane(e.g. positive inside rule), which is also reflected in theasymmetry in lipid composition between inner and outermembrane side. An example for the structural predictionof anArabidpsis sucrose transporter is given in Fig. 2. Thesubstrate recognition is somewhere inside a centralpore, accessible from both sides of the membrane.Though the transporter may have intrinsic rectificationproperties, directionality of transport is not created by thetransporter itself, but the concentration gradients of thesubstrates and coupling mechanism (see below).

Transporters for specific solutes are often similarstructurally and also show surprising conservation at theprimary sequence level among plants, fungi,animals, andbacteria (see e.g. amino acid transport systems, Wipf etal., 2002). Thus they belong to protein families, whichresemble the evolutionary and functional relationshipbetween them. Various classification systems have beendeveloped such as Pfam (Bateman et al., 2002), the SLC

system for human solute carriers ( www.gene.ucl.ac.uk/nomenclature ), and a system comparable to theenzyme nomenclature, i.e. the T numbers (Saier, 2000).The system classifies membrane proteins according tofive parameters: phylogenetic relationship, substrate,transport mechanism and subcellular localization. Since,however, in most cases only the phylogenetic relationshipcan be determined unambiguously and since for mostmembrane proteins the subcellular localization is still notknown, it seems a bit premature to use such a system sowidely. Arabidopsis membrane proteins have beenidentified using prediction programs from the genomesequence, and various databases are available providingan overview and much detailed description of the varioustransporter families (PlantsT: plantst.sdsc.edu, Arame

mnon: Schwacke et al., submitted).

Membrane proteins in complexes and rafts

Like in other organisms, Arabidopsis membrane proteinsare assumed to assemble in homo- or heteromericcomplexes, in clusters or rafts (Reinders et al., 2002b;Jacobson et al., 1995). Recent findings from experimentsusing a variety of biochemical techniques indicate that,although monomers of a transport protein with 12transmembrane spanning domains are mostly sufficientfor the formation of the pore and thus for the transportfunction, most metabolite carriers seem to exist as dimersor even higher order oligomers (Heuberger et al., 2002; forrecent review cf. Veenhoff et al., 2002). Mammalianglucose transporters, although classified as the mostsimple transport systems using facilitated diffusion (seebelow), exist as tetramers, in which the individual subunitscommunicate with each other (Cloherty et al., 2001).Similarly, also the bacterial lacS lactose transporter existsas a dimer in which the subunits interact cooperatively(Veenhoff et al., 2001). Such communication betweenmonomers is thought to be important for controllingtransport activity. Interestingly, a monomer of the lacSlactose transporter functions as a facilitator, whereas thedimer mediates proton-coupled transport (Veenhoff et al.,2001). So far, little is known about such interactionsamongArabidopsis membrane proteins. A main reason iscertainly that the biochemistry of transporters expressedin vascular cells is difficult to study and that classicalmethods for determining protein:protein interactions suchas yeast two hybrid systems were not suitable fordetecting interactions between membrane proteins. Therecent development and application of the split ubiquitin

system to plant membrane proteins provides a new tool todetect and study such interactions (Johnson & Varshavsky1994; Reinders et al., 2002a,b, Schulze et al., 2002). Theplant proton sucrose cotransporters represent distanthomologues of lacS and GLUT1, and are also able to form

oligomers. Three SUT paralogs are present in the samecell and can interact with each other, potentially formingheterooligomers (Reinders et al., 2002b). Also separatelyexpressed halves of one sucrose transporter can interactwith their other half or the half of a paralogous sucrosetransporter leading to the reconstitution of functionaltransporters with new properties. The capacity to interactwith domains of another molecule may suggest thatchimeric transporter pores can be formed within a dimer,providing a potential explanation for the existence of apreferred cooperative interaction between two of foursubunits in a GLUT1 tetramer (Cloherty et al., 2001).

Studies from other organisms have shown thattransporters often form heterooligomers with structurallyunrelated proteins. Classical examples are the bacterial

ABC transporters, which consist of four subunits (Changand Roth, 2001; Martinoia et al., 2002; Locher et al.,2002), whereas in eukaryotes the subunits arepreferentially fused into a single polypeptide. Anotherexample includes the mammalian Na+/K+-ATPase (Kaplan,2002).


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A subset of mammalian amino acid transporters functionas dimers of the catalytic transporter subunit and a heavychain, a polypeptide that contains a single membranespan and a large extracellular domain, which can either berBAT or 4F2hc (Chillaron et al., 2001). The subunit seemsto be required for targeting the transporter itself to theplasma membrane. Furthermore many processes controlthe activity of a given transporter, processes whichrequire interactions with other proteins, e.g. interactionwith the cytoskeleton or with other scaffolding proteins asin case of the human glucose transporter GLUT1 with theraft associated monotopic membrane protein stomatin(Zhang et al., 2001) or with the posttranslationalmodification machinery such as phosphorylation orubiquitylation. Stomatins also interact with othertransporters and channels (Sedensky et al., 2001).

Recent findings indicate that signaling and transportproteins are assembled into large macromolecularcomplexes (Huber et al., 2001; Bickel et al., 2002). In caseof the light perception in rhabdomers, a scaffoldingprotein associates the whole signaling complex includingrhodopsin, Na+- and Ca2+-channels, heterotrimeric G-proteins, phosphokinases and phospholipases into a largecluster (Huber, 2001). Since transport activities at theplasma membrane are not only optimized with respect toaffinity and capacity for single substrates (see belowsensing) but are also involved in the coordination of theactivity between different transporters as in the case ofthe different PII systems in E. coli (Gorke and Rak, 1999)or crosstalk between lactose permease and PII system(Sondej et al., 2002), it is likely that similar tasks must alsobe operational in higher organisms (see below).

It has been suspected for a long time that plasmamembranes contain specific domains and are organized inmosaics (Ikonen et al., 2001, Bagnat et al., 2001; Simons

and Toomre, 2000; Barenholz, 2002). Extraction ofmembrane preparations with the detergent Triton X-100demonstrated enrichment of certain lipids and membraneproteins in the insoluble fraction. Using modified greenfluorescent protein (GFP) halves that are unable todimerize and by attaching lipid anchor attachment sites,the sorting of proteins into rafts could be followed directly(Zacharias et al., 2002). The availability of new membraneprotein purification systems in combination with TAPingand mass spectrometrical analyses will enable analyses ofcomplexes and rafts in much more detail and promises togive novel insights into membrane function and regulationof transport (Ho et al., 2002).

The role of metabolite sensing in regulation oftransport

At first sight, it seems sufficient for a plant to have a singlecarrier for each substrate. Furthermore, if one wouldassume that metabolic reactions are the major

determinants of metabolism, transport might just beconstantly available, and thus does not require significantregulation. However, today it is becoming clear that anorganism uses the full complement of possibilities toacclimate metabolism adequately. Most obvious is thenecessity to regulate uptake of nutrient metals, many ofwhich can be cytotoxic at high concentrations (Connollyet al., 2002). Furthermore external availability of nutrientscan vary significantly. Thus a plant cell will have toacclimate membrane properties by exchanging orregulating transporter activities. Sugar metabolism andtransport seem to be also highly regulated in plants(Hellmann et al., 2000).

Instructive examples of how organisms deal with suchrequirements come from bacteria. Interaction of ferriccitrate with its transporter at the outer membrane inducesa number of conformational changes that finally inducestranscription of its own operon (Braun, 1997; Ferguson etal., 2002). Bacterial PTS transport systems are well knownto provide important information about nutrient availabilityand uptake to regulate metabolism (Saier and Reizer1994; Lengeler and Jahreis, 1996). The phosphorylationstatus is used to monitor the state of the various PTSsugar availabilities in order to hierarchically tuneexpression of the different PTS operons in aphysiologically meaningful way. Thus, the PTS represent ahighly integrated signal transduction network in carboncatabolite control (Gorke and Rak, 1999). Furthermore, theunphosphorylated form of the PTS glucose transportsystem IIAGlc also plays a role in the inhibition oftransport of non-PTS sugars such as lactose, maltose,melibiose, and raffinose (termed inducer exclusion). In thecase of lactose, IIAGlc binds to lac permease to inhibit thelactose transport (Sondej et al., 2002). UDP glucoseuptake into E. coli and other bacteria is controlled by a

pair of highly related proteins, a transporter UhpT and asugar sensor UhpC (Schwoppe et al., 2002). Similarsystems have been described in yeast, where SSY1, anamino acid transporter-like protein is involved in aminoacid sensing at the cell surface and in regulation of aminoacid transporters (Van Belle & Andr, 2001; Forsberg andLjundahl 2001). In case of glucose transport, even twotransporter-like sensors exist which serve as high and lowaffinity monosaccharide sensors and which control themany monosaccharide transporters of yeast in a complexnetwork (zcan & Johnston, 1999; Forsberg and Ljundahl2001; Rolland et al., 2001; Van Belle & Andr, 2001).Similar to yeast sugar sensing, the mammalian glucosetransporter GLUT2 is suspected to serve as a pancreaticglucose sensor (Thorens, 2001). The cell surface is thus

obviously an ideal site to sense nutrients or metabolites inorder to control nutrient uptake. Since the sensors fromyeast are characterized by features such as extendedcytosolic domains for interaction with the signalingmachinery, are weakly expressed and have a low codonbias, one of the sucrose transporters from Arabidopsisand some members of the Arabidopsis monosaccharide


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transporter family have been proposed to serve as plantsugar sensors (Lalonde et al., 1999; Barker et al., 2000).Candidate amino acid sensors are the putative glutamatereceptors, homologs of ionotropic glutamate receptors inanimals (Chiu et al., 1999). Direct proof for the existenceof such sensors in plants is however still lacking.

Inactivation and degradation of transporters

In the context of acclimation of transport, besidesbiogenesis of new transporters also regulated retrievaland degradation of existing transporters is required toprevent that the membrane accumulates too manyunnecessary transporters and to exclude unwantedcompounds from the cell. Cycling of transporters betweenendocytic compartments and the plasma membrane is awell established phenomenon in yeast and mammals(Pelham, 2001). Examples of proteins which may beinactivated by endocytosis and degradation are the yeasturacil permease and the yeast general amino acidpermease (Dupr et al., 2000; De Craene et al., 2001).Phosphorylation and ubiquitination seem to play animportant role in inactivation, endocytosis and cycling ofthese transporters.Also in mammals, transporters are known to cycle.

Glucose transporters such as GLUT4 can be transientlystored in vesicles close to the plasma membrane (Bryantet al., 2002). Upon receiving a signal from insulin, vesiclesare released to the plasma membrane to allow rapiduptake of glucose into the cells (Al-Hasani et al., 2002).On the other hand, rafts seem to be involved insubsequent internalization of GLUT4 (Ros-Baro et al.,2001). Interestingly, GLUT4 interacts physically with

ubiquitin-related proteins involved in degradation andstabilization (Lalioti et al., 2002). Finally, transporteractivity can be regulated directly by physical factors ase.g. in case of transporters involved in osmoregulation(Poolman et al., 2002).

In plants much less is known about regulation oftransporters at the posttranslational level and on cycling.As an example, sucrose transport activity is affected bysucrose and sucrose transporters may be regulated byphosphorylation (Chiou and Bush, 1998; Roblin et al.,1998). Furthermore the sucrose transporter SUT1 isturned over very rapidly, suggesting that activemechanisms are also involved which provide a means tocontrol sucrose transporter activity also by degradation(Khn et al., 1997).

How transporters are targeted to their destinedcellular location

As cellular compartments have different functions, so do

the membrane proteins differ within membranes delimitingthese organelles. Each protein is targeted to therespective membrane by a specific address label (ZIPcode) to allow delivery to the correct compartment, whilesome proteins may be targeted to more than one organell(Peeters and Small, 2001). In some specialized cases,there maybe even protein transport to adjacent cells, thusthe cell-cell connecting plasmodesmata have to recognizespecific ZIP codes.

Plasma membrane proteins contain specific informationthat directs their targeting via ER and Golgi to theirdestination in the plasma membrane (Goder and Spiess,2001; Pelham, 2001). Briefly, a hydrophobic signalsequence in a polytopic membrane protein may serve as asignal for the signal recognition particle (SRP). Afterinitiation of translation upon ribosome binding to thetranscript, the SRP/ribosome/nascent chain complex(SRP-RNC) docks with the SRP receptor on thecytoplasmic face of the ER. Thereafter, eachtransmembrane domain (TMD) is translated on theribosome and may be inserted cotranslationally into theER membrane. Concurrently and/or subsequently, one ormore transmembrane domains at a time presumablymove laterally to the periphery of the translocon and intothe bilayer. From there the membrane protein traffics tothe Golgi. Loading of the vesicles may involve cargospecifiers (Gilstring et al., 1999; Antonny and Schekman,2001). Within the Golgi, proteins are either sorted intovesicles destined to the plasma membrane or to thevacuole (Pelham, 2001). Thus membrane proteins use apathway similar to those of secreted soluble proteins, butthey always remain integrated into the membranes toreach their target membrane. However, in contrast tosoluble protein ZIPs, which are normally cleaved withinthe endoplasmic reticulum (ER), the signal sequences ofpolytopic membrane proteins are usually not cleaved. It isoften assumed that the N-terminus and/or firsttransmembrane spans serve as ZIP codes (for review cf.Chin et al., 2002).

Since ER import of transmembrane proteins is related tomembrane sorting in bacteria, one may use the wealth ofinformation generated to extrapolate from knowledgeavailable for E. coli transporters. Interestingly, the lengthof the central loop of lac permease seems crucial forcorrect integration of the transmembrane helices into themembrane (Weinglass & Kaback 2000). The functionalreconstitution of separately expressed sucrose transporterhalves in yeast suggests that both halves of the sucrosetransporters contain signal sequence information to enterthe ER. Since, in ER-targeted soluble proteins, the

cleavage product provides experimental access to identifyand characterize signal sequences, ZIP codes of solubleproteins are far better understood as compared tomembrane proteins.

Various algorithms and programs have been developedthat allow prediction of the destination of a query protein(Schwacke et al., submitted). However the prediction for


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membrane proteins so far is not very reliable (Bannai etal., 2002; Emanuelsson et al., 2000; Schwacke et al.,submitted). Proteins destined for the tonoplast must alsouse specific signal sequences for sorting into vesiclesfusing with the vacuole. However, a detailed comparisonof the sequencewise highly related tonoplast and plasmamembrane aquaporins has not yet allowed theidentification of such signal sequences (Johanson et al.,2001). Membrane proteins that transport solutes acrossorganellar membranes also use ZIP codes forposttranslational sorting. In the case of the prototype ofplastidic transporters, the triosephosphate translocator(TPT), a cleaved signal sequence has been identified(Flgge et al., 1989). Interestingly, a member of theplasma membrane hexose transporter family isresponsible for plastidic glucose transport and contains asignal similar to that of the triose-phosphate translocatorfor targeting to plastids (Weber et al., 2000). Similarly, assulfur metabolism is predominantly found in chloroplasts,a member of the plasma membrane sulfate transporterfamily containing a signal sequence has been localized toplastids (Takahashi et al., 1999). Some inner mitochondrialmembrane proteins also have a clear signal sequence,while others, although known to be localized to thesemembranes, do not contain obvious signal sequences. Todate, reliable information about the subcellular localizationof proteins can only be obtained experimentally. However,even the available methods may be erroneous due tomistargeting upon overexpression or fused reportermolecules (Okumoto et al., unpublished). So far, very littleis known about these targeting sequences, althoughprediction programs can give an idea about subcellularlocalization (see for example Schwacke et al., submitted).

Transporters for a wide range of substances

Within living species, specific transporters for probably allclasses of substances exist many can serve as nutrientsfor Arabidopsis, suggesting that a wide spectrum ofcarriers on the root surface and rhizodermis exists. Manyplants form a symbiosis with mycorrhiza, but not

Arabidopsis. This plant must acquire all these nutrients onits own. Therefore it is an ideal model system tounderstand principles of nutrient uptake. In order toevaluate the function of a plant transporter, the substratespecificity is of prime importance. In addition, theintracellular localization (plasma or organellar membrane)and cell-type specificity within a tissue determine its

function. Expression in the vasculature suggests afunction in long distance transport and in partitioningbetween tissues of supply (sources) and tissues ofdemand (sinks). Although historically plant membranetransport has often been investigated in species otherthan Arabidopsis, the ease of identification of mutants,completion of the genome sequence, the many genetic

and molecular tools available, and a large scientificcommunity promise that Arabidopsis will be the plant touncover many fundamental plant transport processes inthe future. For a general reading of plant membranetransport, students are recommended to read therespective chapters in textbooks as Taiz and Zeiger (1998)or Buchanan, Gruissem and Jones (2000).

General furnishing with proteins in every higher plantmay be similar, and the specificity of a given plant mayarise from specific regulation of a small set of genes. Forexample, in some legumes like pea and bean, besidesamines or amino acids, ureides are a major transport formof fixed nitrogen in the phloem. Ureide transporters arealso found in the genome ofArabidopsis (Desimone et al.,2002), although ureides have not been detected in thephloem sap so far. Some plants, however, clearly havedistinct transporter types, as the LCT1 calcium transporterfrom wheat has no homologs inArabidopsis (Schachtmanet al., 1997). Transport proteins for prominent solutes aremembers of multigene families. The reason for theapparent redundancy is not clear, but fine tuning isprobably involved. It can be expected (and has beenshown for amino acid transporters (AAPs) and sugartransporters SUTs) that many transporters haveoverlapping substrate specificities. Due to the methodshow the transporters were identified, the true substrate (orsome of the substrates) in the plant may not have beenidentified. The competition of amino acids on binding toamino acid transporters has been shown for AAPsexpressed in yeast (in growth assays) and directlyexpressed in oocytes (Fischer et al., 2002).

Imaging of metabolites in Arabidopsis

Compartmentation of metabolic reactions and thustransport within and between cells can only beunderstood if their subcellular distribution has beendeternined by non-destructive dynamic monitoringtechniques. Currently, methods are not available for invivo metabolite imaging at cellular or subcellular levels.Limited information derives from techniques requiringfixation or fractionation of tissue (Farr et al., 2001;Borisjuk, 2002). Such static analysis of metabolitecomposition in organs, tissues and cellular compartmentsinvolves cell disruption. Thus most techniques neithermeasure metabolite changes in real-time nor account forlikely variation in local metabolite concentration at thecellular level. Furthermore, these methods have low

resolution and are prone to artefacts, e.g. contaminationby other cell types or subcellular compartments. Little isknown about the dynamic changes in concentration ofmetabolites such as sugars and amino acids at the site oftransport, i.e. at the loading site of the phloem in plants,but also concerning the distribution of different sugarswithin cellular subcompartments. To better understand


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metabolism and compartmentation, non-invasivetechniques would be of significant advantage, such as theFRET (fluorescence resonance energy transfer) system toimage calcium fluctuations (for review cf. Tsien 1998).Using a similar approach, novel nanosensors for sugarswere developed by transforming substrate-induced hinge-bend movements upon substrate binding into changes influorescence resonance energy transfer between twodifferent fluorescent proteins (YFP and CFP; Fehr et al.,2002). The backbone of the nanosensors are bacterialperiplasmic solute binding proteins. The nanosensorswere shown to function in vitro and detect sugars incomplex mixtures. Furthermore, uptake of sugars intoyeast cells expressing the nanosensors in the cytosol wasvisualized in real time. Given that periplasmic bindingproteins exist for a wide spectrum of substrates, suchstrategy may provide a basis for the development of avariety of transgenic plants for analysis of ion andmetabolite concentrations, transport and sensing.

Different organs of Arabidopsis are connected byvasculature

Many nutrients and metabolites such as sugars, sugaralcohols, amino acids, organic acids and nucleotides aretranslocated between different organs within plantspreferentially in the vasculature. In addition, hormonesand some secondary metabolites are also transportedbetween organs via these conduits. Plants including

Arabidopsis use two major routes for long distancetransport, i.e. the phloem and the xylem (see below),which can be compared to the highways for long distancetransport. On top, slower country roads exist, that allow

long distance transport by cell-to-cell movement (e.g.polar auxin transport in the xylem parenchyma). Many ofthese compounds may be imported into the vascularsystems via specific transporters, while others may be co-transported in concert with other substances by the sametransporter. Metabolite profiling of phloem and xylemexudates will lead to an expansion of our view of whichsolutes are transported in the vascular saps (Fiehn et al.,2000). Many of these transported substances may haveboth nutrient and signal function. The presence of aspecific set of transporters thus controls whichsubstances can move in the phloem or xylem saps. Thustransporters allow uptake of sucrose, but not glucose intothe phloem stream. Nevertheless, several studies haveindicated that the transport systems involved have a

limited selectivity for their substrates. Amino acidtransporters transport a wide spectrum of different aminoacids and derivatives (Fischer et al., 2002). Sucrosetransporters transport phenylglucosides and even biotin(Ludwig et al., 2000). Thus many xenobiotics may be ableto traffic in the plant by means of such low-selectivetransport systems. The extended selectivity for the

substrate may even allow to design large substrateconjugated molecules that act as xenobiotics which areimported into the vascular system (Deletage-Grandon etal., 2001).

Para- and transcellular pathways for solute transport

Arabidopsis as an organism composed of a wide varietyof cell types does not only require transport systemsallowing uptake and release of compounds into and fromindividual cells, but in addition has to exchange solutesbetween cells. This becomes most apparent whenconsidering the specific requirements of different organssuch as roots and leaves. Leaves produce assimilatessuch as sucrose, whereas all the essential mineralnutrients are almost exclusively taken up by the root. Thusboth systems are fully dependent on exchange of solutesvia the vascular systems. But even within an organ,different cells types fulfil different functions and evenbiosynthetic pathways may be compartmentalizedbetween different cell types within the same tissue (St-Pierre et al., 1999). Two principal routes exist for soluteexchange between neighboring cells: (i) the paracellularpathway, in which solutes pass through the cell wall,

Figure 3. Schematic view of para- and transcellular solute flow.During paracellular solute flux in the aqueous cell wall space, theapoplasm, solutes neither cross membranes, nor enter the cells.Transcellular solute movement is characterized by initial soluteinflux into the cell. Further flux may include efflux into theapoplasm and subsequent reuptake by the neighbouring cell orsymplasmic movement.


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a space named apoplasm and (ii) the transcellularpathway where solutes move via the cytoplasm, definedas the symplasm (Fig. 3).

Intercellular translocation can occur by movementthrough the apoplasm, as long as the continuity of cellwalls is not interrupted by barriers such ascutinized/suberinized layers, as e.g. in case of theCasparian strip, which separates the rhizodermis from thestele. Xylem vessels are formed by dead cells thus theybelong to the apoplasm since they are continuous withthe cell wall space and are not separated by a membranefrom each other. Thus translocation in the apoplasm canhave a direction and it can be much more rapid thandiffusion if water moves due to root pressure, capillarypressure, transpiration or other factors, e.g. in the leafapoplasm. Directionality is given here by the direction ofwater flux through the plant. In case of transcellularmovement via the symplasm, translocation occurs via theplasmodesmata, which are highly specialized intercellularconnections not found in animals. Almost all Arabidopsiscells are interconnected by plasmodesmata. The best-known exceptions are guard cells, in whichplasmodesmatal connections are eliminated duringdevelopment. Plasmodesmatal structure and function stillis only poorly understood. So far, no direct evidence hasbeen presented for movement of small solutes throughplasmodesmata. However chemical compounds such asdyes, but also RNA, proteins and even virus can movebetween cells, we generally assume that small moleculescan also freely be exchanged between neighboring cellseither through the desmotubules or through the spacebetween plasma membrane and ER strands within aplasmodesma. If the hypothesis of free movement of ionsand other small solutes through plasmodesmata holdstrue, the availability of open plasmodesmatal

macrochannels would have important implications: themembrane potential can not be regulated by individualcells, energization is delocalized. The H+-ATPase activityin one cell may energize a process taking place in theadjacent cell. Solutes may move simply by diffusion fromcell to cell, along gradients established by metabolicreactions in different cells connected by plasmodesmata.This provides a unique pathway through what is called thesymplasm, since the individual cytoplasm of the differentcells is interconnected. Obtaining direct proof forsymplasmic transport of solutes is complicated due to theexistence of cell walls and the complexity ofplasmodesmata. Double patch clamp studies similar tothose used for analysis of mammalian gap junction andnovel imaging techniques are required to prove directly

that small molecules can move freely. The relative role ofsym- and apoplasm routes is obviously also important forquestions relating to long distance translocation of soluteswithin a plant.

Long distance translocation in the vasculature

Nutrients may be taken up directly at the root surface, e.g.into the root tip cells or root hairs and then movesymplastically or may move in the apoplasm up to thesuberinized endodermis, which in Arabidopsis is locatedonly two layers below the rhizodermis (Barton 2001). Afterentering the central cylinder, inorganic ions as well asorganic molecules have to be exported into the apoplasmnear the xylem vessels in order to be transported to theshoot. The vectorial transport from root to shoot is notonly due to the driving forces acting on xylemtranslocation, but also to cell specific expression ofcarriers, e.g. in xylem parenchyma. The transport pathwayfor nutrient redistribution to plant parts distant from thesoil is the xylem, the main long distance transportpathway for mineral nutrients and water. Flow in the xylemis essentially unidirectional, from root tips to the sites ofwater evaporation.

The driving forces, however, are not yet fully understood(Beevers and Tanner, 2001). Although consisting mainly ofdead cells, the xylem contains living xylem parenchymathat is probably not only involved in generating drivingforces for xylem translocation, but also plays an importantrole in regulating flux (Zwieniecki et al., 2001). The watertransported in the xylem is either evaporated via stomata,secreted via hydathodes or recycled via the phloem. Thehydathodes in leaves are connected to open ended xylemvessels, covered by a cap structure called epithem.Secretion occurs through specialized stomata.Hydathodes are related to nectaries and thus aresupposed to be able to secrete specific substances whileretaining others. In broad terms hydathodes can thus becompared to kidneys that must contain a specific set of

carriers to fulfill these retrieval functions. Growing andexpanding leaves, as well reproductive organs that arelow in transpiration receive little supply by the xylem.Similarly, optimal supply of photoassimilates to storageand developing tissues is provided by long distancetranslocation in the phloem. However, since the solutemovement in the phloem is driven by the sites of soluteproduction (sources) and utilization (the sinks), thedirection is under continuous changes driven by thedifferent demands during development (Busse and Evert,1999a,b). The actual conduits are the sieve elements,peculiar cells lacking a nucleus in the mature stage andtightly associated with companion cells (Van Bel andKnoblauch, 2001; Van Bel et al., 2002; Lalonde et al.,2001). An instructive example is the expression of a

sucrose transporter during transition of a leaf from sink tosource (Fig. 4).The vascular bundle holds four to more than 20

independent sieve elements in parallel, thus movement


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Figure 4.-Glucuronidase assay of the localization of promoteractivity of the source specific AtSUC2, a companion cell specificsucrose transporter. The picture shows promoter activity invascular bundles of leaves, stems and roots. Cotyledons (leaf No.1,2) GUS staining follows the transition from sink to source and isstrongest in old leaves and increases in young leaves from bottomto top. (Courtesy by W. Schulze)

within a vascular bundle can be uni- and bidirectional, butdue to the general rule from high pressure to lowpressure, flow cannot be bi-directional in the same sieveelement. Though transport may be mediated by osmoticgenerated pressure flow (OGPF), the specificity has to beestablished by specific transporters, which again have tobe expressed in a cell specific manner. The definition ofsink and source status thus is defined by the major

osmolytes that enter the phloem, mostly by activetransport. Since in many plants the major osmolyte issucrose, followed by amino acids and potassium,directionality is created by the loading and unloading ofthese substances. Therefore the terminology is usedpreferentially in the context of sugar movement, whereasin case of other solutes, e.g. amino acids, the terminologyof sink and source is less useful, especially sincemovement occurs in phloem and xylem and directionalityof amino acid translocation is controlled by differentmechanisms.

Sugars, which are produced by photosynthesis mustmove from the sites of synthesis, i.e. the mesophyll to thephloem. Theoretically, movement to the phloem can besymplasmic, as 1-10 plasmodesmata/m-2 connect

mesophyll cells with the companion cell/sieve elementcomplex (Haritatos et al., 2000). This plasmodesmatalfrequency and the lack of distinct intermediary cellmorphology in minor vein companion cells classifies

Arabidopsis as a type 1-2a species (Gamalei, 1991).Morphologically, Arabidopsis represents thus anintermediate species between typical type 1 symplasmic

loaders and pure apoplasmic loaders (type 2). Asexpected for a type 1-2a species, the main transportsugar in Arabidopsis is sucrose, but also raffinose andtraces of galactinol are transported (Haritatos et al., 2000).

Arabidopsis may therefore be an excellent model systemto analyze the putative contribution of symplasmic loadingand raffinose transport in addition to sucrose transport inlong distance translocation. In contrast to themorphological data, the phenotype of mutant plants witha deletion of a single companion cell sucrose transporter,AtSUC2, classifies Arabidopsis as an apoplasmic loader(Gottwald et al., 1999). Complete knock-out of thetransporter gene resulted in stunted growth, retardeddevelopment and sterility. Source leaves contained greatexcess of starch, and sugars were only minimallytransported to sink organs as roots and inflorescences(Gottwald et al., 1999). The AtSUC2 transporter isexpressed in the phloem of source tissues (Truernit andSauer, 1995; Fig. 4). It is expressed in major veins inleaves that act as sinks, and in all veins when a leaf is aclear exporter of sucrose. A gradual transition from asucrose importing leaf located at the tip to a sucroseexporting leaf at the base can be followed by equivalentexpression of SUC2 (Fig. 4). For sucrose, a concentrationgradient is created by proton-coupled sucrose transportat the sieve element/companion cell complex (SECC, seebelow). The concentration of sucrose (and otherosmolytes) in the phloem sap creates water potentialleading to water influx from the surrounding tissue,including the xylem. This results in a positive pressure inthe phloem. Translocation is driven by mass flow ifsucrose is unloaded in the sink organs. Only little efflux isobserved along the path by using phloem mobile dyes(Wright and Oparka, 1997; Imlau et al., 1999). In mostcases, sieve element unloading is assumed to proceed via

bulk flow through plasmodesmata. Because of the largepressure gradient involved, this first step is essentiallyirreversible. The phloem is unloaded in root tips, andunloading of carboxyfluorescein and green fluorescentprotein from jellyfish suggests that the size of theplasmodesmatal pores connectingArabidopsis plant cellshave size exclusion limits larger than the typical 800Da,suggesting greater conductivity, but the size exclusionlimit for plasmodesmata may vary upon environmentalstimuli. Uptake into seeds requires apoplasmic transportsteps since endosperm and embryo are symplasmicallyisolated. Both cellular exporters and import systems in theendosperm and the cotyledons of the embryo contributeto phloem unloading (Roche et al., 2002). The physiologyof unloading processes has been described elsewhere in

detail (Patrick and Offler, 2001; Lalonde et al., 2002).

Hormones

Plant hormones ( auxin, cytokinin, ethylen, abscisic acid,


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jasmonic acid, giberrellin, brassinosteroids, peptides,salicylic acid) play a key role in controlling the growth anddevelopment of plants. Plant hormones are organiccompounds synthesized in one part of the plant and inmost cases are translocated to another organ. Theygenerally act at very low concentrations (often


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Figure 5. Schematic representation of transport mechanisms anddirection of transport. Left side outside, right side inside.

not necessarily mean that a substance is moving fromhigher to lower concentration, as the sum of both theelectrical and the chemical term add up to theelectrochemical potential. Active transport is used todescribe the uphill movement of a substance against itselectrochemical potential.Primary active transporters couple light energy or the

energy released upon hydrolysis of a chemical bond tothe conformational change of a membrane protein, and bythat couple transport of a substance against itselectrochemical potential. The P-type H+- and metalpumps, the organellar proton pumps and many ABCproteins belong to that class.Secondary active transporters use the free energy

difference stored in the electrochemical gradient ofprotons or other solutes across the membrane to drivesimultaneous movement across the membrane of anothersubstance. In effect this energetically adds up to a netdownhill flow. Depending on the direction of transport andwhether a coupling ion is used, symport and antiport arediscriminated. Such transporters are symporters (oftenalso simply considered as co-transporters) when two

dissimilar solutes (substrates) are transported together orsequentially within a transport cycle into the samedirection across the membrane. Transport of the twosolutes is obligatorily coupled. In vivo, secondary activeplant co-transporters are generally H+-coupled systems,so that the large electrochemical potential for protons,generated by primary active transporters, is coupled tothe uphill movement of another substance. The transportis considered as antiport, when the direction of bothsubstances is opposite. The conformational changesassociated with the transport do not require hydrolysis ofchemical bonds, but solely rely on the electrochemicalgradients for both substrates.

Examples of passive transporters are channels, likewater or potassium channels. Other passive transportersare uniporters that transport only one substance passivelydownhill an electrochemical gradient. Although thedistinction may be arbitrary, membrane passage throughuniporters or carriers is often assumed to involveconformational changes within the transport cycle, oreven diffusion of the carrier with bound or withoutsubstrate through the lipid membrane, while channelmediated transport does not involve conformationalrearrangements, although channels may open and close.

Polarity of transport processes

It is obvious that transport is a vectorial process. In amulticellular organism, solutes have to be transportedfrom one cell type or organ to the other. Directionality canonly be achieved if mechanisms are at hand to drivetransport in a certain direction. Besides polarity within atransport system (e.g. by coupling to a defined gradient,

i.e. proton gradient), it is essential that transporters areexpressed only in a certain cell type. For example, thecoexistence of cotransporters for the same substrate inadjacent cells will lead to competition between the cellsrather than directional transport. In contrast, if only one ofthe two cells expresses the transporter gene, whereas theother contains a uniporter or an exporter (e.g. a protonantiporter), flux will be directed from one cell to the other.Therefore many transporters have highly cell-specificexpression patterns. This also means that in order toincrease flux, it is not possible to overexpress thetransporters under control of general cell-non-specificpromoters like the CaMV 35S promoter as is normallydone for increasing enzyme activities, since this shouldcollapse directionality and thus may be counter-

productive. For transcellular transport, as e.g. inmicrovillar cells in the mammalian intestine, uptake andrelease carriers may be transported to specific domains inthe polarized cell. Provided that at least one of the carriersis primary or secondary active, this polarization allowsunidirectional flux across the cell. A typical example forplants is the polarized distribution of auxin influx and


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be expressed under control of a yeast promoter, and inthe simplest case, yeast mutant growth is monitoredunder selective conditions. Selective conditions may bee.g. media without the substance that the yeast isauxotroph for. Only if the transfected cDNA provides themessage to express a transport protein that amelioratesthe auxotrophy, yeast can grow, and the cDNA carryingplasmid is isolated and sequenced (Fig.6). This approachhas the enormous advantage that no other information isnecessary about the transporter, and the isolated cDNAautomatically encodes a functional transporter by itself.This method has been used to isolate plasma membraneand organellar transporters. Functional complementationof simpler systems as bacteria has not been assuccessful, possibly due to toxic effects of heterologousmembrane protein expression on E.coligrowth (Frommerand Ninnemann, 1995). However, mammalian COS cellshave also been used to identify heterologously expressedplant aquaporins (Kammerloher et al., 1994). The yeastcomplementation system identifies thus transporters for agiven substrate, but if this substrate is indeed the truesubstrate within the plant is not known. In addition, onlytransporters that are functional as single polypeptides willbe isolated by that approach, but some plant transportersmay be encoded by multisubunit complexes (see above).Transporters may even be false targeted to othermembranes in heterologous systems, thus a plasmamembrane transporter in yeast may not necessarilyencode a plasma membrane transporter in plants.

efflux carriers permitting polarized transcellular hormonefluxes (Friml and Palme, 2002).

Molecular identification of transporters

Our knowledge about the true substrate of a given planttransporter is often limited. For example, transporters foramino acids often transport many different amino acids,and according to the availability, the true substrate in theplant may differ from the substrates that the transporterwas initially isolated for. Sucrose transporters are able totransport additionally other glucosides and evenstructurally unrelated biotin. Although sucrose transportmay quantitatively be the pivotal function, transportersmay be involved also in transport of many othersubstances. Therefore, the widespread different strategiesused to identify transporters will be shown andadvantages and limitations will be briefly discussed.

The most widely used and probably most successfulapproach to identify plant transporters for a givensubstrate is suppression cloning in heterologous hosts,especially yeast (for reviews cf. Frommer and Ninnemann1995; Barbier-Brygoo et al., 2001). The basis of thisapproach is a yeast lacking a specific transport capacity,either because yeast does not have such a system orbecause the endogenous system has been inactivated,e.g. by supply of a suppressing agent (ammonium forgeneral amino acid permease GAP1), or by mutating (inmost cases deleting by homologous recombination) thetransporter gene(s) or by manipulating the strain in a waythat it becomes dependent on an uptake system. Thisstrain is transformed with a plant cDNA library, which can

Many of the advantages and disadvantages also apply toa related approach: expression cloning in frog oocytes.Here radiotracer uptake or ionic conductances induced byexpression of plant mRNAs are monitored in singleoocytes, and starting from simultaneous injection of awhole library into single oocytes, the activity induced by a

single cDNA clone is isolated. This approach has beenextremely successful for identification of mammalian ionchannels and transporters, but was less widely used forplant transporters, although many plant transporters canin principle be expressed in oocytes (Boorer et al.,1996ab; Zhou et al., 1997; 1998; Fischer et al., 2002;Desimone et al., 2002). This approach has so far onlyyielded the cloning of a syntaxin, which may indirectlyaffect endogenous ion channels in the oocytes (Leyman etal., 1999).Another highly successful method to isolate transporters

is the screen of plant mutants involved in specifictransport tasks within the plant. Starting from EMSmutagenized or T-DNA tagged lines, morphologicalmutants were often easily identifiable as being involved in

plant hormone transport. A disadvantage of this approachis that, as transporters are often members of multigenefamilies, other transporters may compensate for the lossof a specific transporter. This approach may be especiallypromising with plant hormone transporters, since alteredtransport of hormones may lead to easily observablephenotypes.

Figure 6. Representation of a functional complementation

approach to identify plant transporters for specific substrates.mRNA has to be extracted from the plant, and cDNA issynthesized by RT-PCR. Size fractionated cDNAs are ligated intoa yeast expression vector. Plasmids are transformed into yeast,e.g. a histidine auxotroph (JT16) mutant. Growth on selectivemedia allows only yeast to grow that express a transporter orenzyme that circumvents the auxotrophy. After re-isolation of theplasmid the encoding cDNA is sequenced.


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Among the advantages of that method is that it directlydefines the functional role within the plant, as the loss isalready observable in the tissue where the transporter isnormally expressed. However, in a complicated systemsuch as the multicellular plant, a regulatory effect on atransporter can often not be excluded. In the absence oftransport function in a heterologous system or whenbiochemically isolated, these proteins have to beconsidered as putative transporters.A strategy that currently is widely used with the

sequencing and analysis of other related organisms, iscandidate cloning by homology. This approach is simple,but needs a lucky guess on the substrate andsubcellular localization. Other methods, like differentialscreening of a cDNA library have been used (e.g. foridentification of a algal sugar transporter HUP1; Sauer etal., 1989), or labelling by specific substrate or inhibitorbinding, purification, reconstitution, sequencing of protein(Flgge et al., 1989). If a highly abundant transporterrecognizes highly specifically its substrate, such astrategy may be advantageous, but may fail due to manyexperimental difficulties. Thus nearly in all approaches themethods may not identify the true substrates, even if onesubstrate is identified.

For a given nutrient, often multiple transport systemscoexist within a single cell to assure uptake over a broadrange of substrate concentrations. These systems arefrequently referred to as high and low affinity systems.The quantitative parameter is the Km that determines theaffinity of the substrate concentration at which thetransport flux (Jmax) is half-maximal (in analogy to enzymekinetics). Similar to enzymes, many transporters haveaffinities in the millimolar and micromolar range. The Kmoften reflects the order of magnitude of substrate thetransporter is facing. This has the advantage that thetransporter is not saturated, and the substrateconcentration can easily be kept constant. If it would besaturated all the time a little bit more would lead toaccumulation, if the affinity of the transporter would bemuch lower than substrate concentration, accumulationwould be much less effective, especially in thecompetition with another binding site. Binding sites ofdifferent organisms might compete about substrates.

Using mainly suppression cloning in yeast, of the ~6475membrane proteins from Arabidopsis, about 50% havebeen assigned a putative function. The rest is unknownand classified as hypothetical protein (Schwacke et al.,submitted).

Proton pumps

Directionality in transport is not only due to differentialexpression of transporter proteins, but may also be due todirectionality of the transport process itself. Membranesare not only used to enclose compartments, but also to

generate potentials that determine which compoundsmove in which direction. Arabidopsis uses mainly protongradients to generate membrane potential (Sze et al.,1999). This potential sums up from an electrical and achemical component. Cell walls are therefore often acidic,and the membrane potential is in the range between -120and -250mV. These two components can be used to drivetransport of other compounds in a certain direction, e.g.into the cell in a concentrative manner. The keycomponents of the cellular machinery responsible forcreating these gradients are pumps that extrude protonsinto the cell wall space or into the vacuole.Active transmembrane proton transport is mediated by

proteins that couple the energy released by cleaving ahigh energetic bond, e.g in ATP, to conformationalchanges in a protein (Sze et al., 1999). Thisconformational change then mediates uphill transport ofthe substrate. With the exception of the pyrophosphatase,all plant pumps share homologs or orthologs in alldomains of life, including humans. This is also true forseveral passive transporters and underscores how thefundamental transport processes are similar in allorganisms.

P-type pumps

These pumps derive their name from the presence of acovalent pump-phosphate transition state during thereaction cycle that distinguishes P-type from F-typeATPases. During the reaction cycle, the -phosphate ofATP becomes transiently, but covalently, bound to acytoplasmic aspartyl residue. While H+-binding sites withinthe protein are exposed to alternate sides of the

membrane, the hydrolysis of this acyl-phosphate bondsupplies the driving force for the conformational changethat translocates one cytoplasmically bound protonagainst its driving force. Later protein rearrangementslower the external proton binding site affinity and permitH+ dissociation. P-type ATPases are specifically blockedby the structural analog orthovanadate.

P-type pumps have typically 10 TMs, a molecular mass100 kDa and are encoded by 45 genes in Arabidopsis.They may be grouped into 5 subfamilies. Severalmembers of a subgroup of 12 proteins (P3A) have beenfunctionally shown to encode plasma membrane H+-pumps. An overview can be found in Axelsen andPalmgren (2001). Other subfamilies are heavy metalpumps (P(1B), 7 members), calcium pumps (P(2A) and

P(2B), 14 members). The last subgroup (P(4), 12members) has been implicated in aminophosholipidflipping, but this may be an indirect effect of pumpactivity. P-type pumps do not necessarily localize to theplasma membrane. All P-type pumps actively transportthe substrate uphill, from the cytoplasm into the apoplasmor organelles.


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Vacuolar, mitochondrial and chloroplastic H+-pumps

The vacuolar-type (V-type) proton pump is localized bothon the tonoplast and in endomembranes. It activelypumps protons from the cytoplasm into theendomembrane lumen, like the endoplasmatic reticulum,into Golgi vesicles and into the vacuole (Sze et al., 2002).The pump is a multisubunit complex that is composed oftwo functionally distinct major components: themembrane integral Vo that mediates the protontranslocation, and the cytoplasmically oriented hydrophilicheadgroup V1 that mediates ATP-hydrolysis. Bothcomponents are connected and stabilized by additionalsubunits, but are easily dis- and reassembledin vitro. Thefunctional significance of the de- and reassembly is notyet fully clear. The membrane intrinsic part is composedof multiple copies of the extremely hydrophobic subunit c(therefore called proteolipid). These subunits form atransmembrane pore for H+-translocation. The center ofthe pore is filled by the subunit, which is also bound tothe center of V1. The headgroup is mainly composed of athree-fold alternating arrangement of - and - subunits.Reversible and ordered binding, ATP-hydroplysis andADP release at the three binding sites for nucleotidesdrives rotation of the central -subunit and transport of H+in the pore. Normally two H+ are translocated perhydrolysed ATP, but the ratio may be influenced by theluminal pH. Several subunits are represented only once inthe Arabidopsis genome, while others are identified withmultiple, exchangable copies. The proton "pump" foundat the chloroplastic thylakoid membrane, the CF-type ATPsynthase, is a distant relative of the V-type ATPase.Although structurally related, the in vivo function isessentially reverse (inverse) to that of the vacuolar pump:

the CF-type ATP synthase generates ATP from ADP andPi, by using the energy stored in the proton gradientacross the thylakoid membrane. Again, the pump hassimilar multisubunit structure and is composed of ahydrophilic ATP-hydrolyzing head group (F1) and thehydrophobic proton channel (F0). The photosyntheticproton accumulation in the thylakoid lumen, induced bythe light-stimulated electron transport chain, develops alarge proton and electrical gradient across the thylakoidmembrane. The protons passively flow downhill from thestroma according to their electrochemical potentialthrough the proton channels conferred by the CF-typeATPase. During H+-flow, they induce a rotary movement ofthe central subunit of the proton pump and drive theconformational changes responsible for ATP-synthesis.

A similar proton motive force is established across theinner mitochondrial membrane. However, it is not lightinduced, but redox-potential driven. In that membrane, adistinct but structurally similar F-type pump acts as anATP-synthase. Thus, physiologically both proton pumpsact in a reverse mode. They synthesize one ATP pertranslocation of 3-4 protons. The reaction cycle of the

chloroplastic and mitochondrial pump is, in contrast to thevacuolar pump, reversible, thus ATP hydrolysis canestablish a proton gradient across the respectivemembranes. Though specific parts of the pumps arehomologous, the genes encoding plastidic proton pumpsare distinct from the endomembrane pump.

Pyrophosphatase

Another H+-pump (H+-PPase) that couples hydrolysis ofinorganic pyrophosphate to proton translocation is foundspecifically in vacuoles, but probably not inendomembranes. Homologs are encountered only inplanta, microorganisms, archaebacteria and bacteria. Incontrast to the V-type ATPase, the H+-PPase is composedof a single polypeptide of 85 kDa with 14-16 TMs, andthe active form is thought to be a homodimer.

Pyrophosphate is an unwanted by-product of severalbiochemical pathways involved in cellulose, nucleic acidand protein metabolism that are especially active inimmature tissues. In these tissues the H+-PPase may bemore active than the V-type ATPase. Thus the H+-PPasemay function to hydrolyze excess PP i and store thereleased energy in a large vacuolar proton gradient. TheH+-ATPase is represented by 3 members in the Arabido-

psis genome.

ABC transporters

In plants many secondary, often plant specificmetabolites, like flavonoids, anthocyanins or breakdown

products of chlorophyll are sequestered into vacuoles,often for defense purposes. In addition, vacuolarsequestration serves as a mechanism to detoxify thecytoplasm from xenobiotics-synthetic compounds suchas herbicides. Vesicles made from vacuoles can importthese compounds in an ATP-dependent manner, but thisimport is not sensitive to protonophores that dissipate thevacuolar proton gradient. Thus this transport, althoughactive and energy requiring, is independent of thevacuolar H+-pumps and the proton motive force acrossthe tonoplast. Transport is not sensitive to V-type pumpinhibitors like bafilomycin, but is rather sensitive tovalinomycin.

The molecules that mediate the transport of such diversesubstances by coupling the release of chemical energy

stored in ATP to substrate translocation are called ATP-binding cassette (ABC) proteins. Although manyhomologs of this class of proteins are found in bacteria,archeabacteria, yeast and mammals, the microscopicdetails of substrate recognition, transport mechanism andenergy coupling are still almost obscure. Only a few planttransporters and their substrate specificities have been


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analysed in detail (Martnoia et al., 2002). The ABC-transporter class is known for an extraordinary broadsubstrate spectrum: in mammals and yeast some ABC-transporters confer "multidrug resistance" by exportingstucturally diverse drugs and peptides, others transportlipids or heavy metals in a phytochelatin-dependentmanner. The human homolog CFTR (cystic fibrosistransmembrane regulator) is a chloride channel, whileanother ABC-protein, the sulfonylurea receptor, acts as aregulator of ion fluxes, by specifically regulating apotassium channel. In bacteria, some ABC-transportersimport amino acids or mineral nutrients.At least four plant ABC-transporters (AtMRP1-3,5)

function in vacuolar sequestration of flavonoids andxenobiotics after their conjugation with the thiol-tripeptideglutathione: they are localized to the tonoplast and act asglutathione S-conjugate pumps (Martnoia et al., 2002).AtMRP2 additionally transports linear tetrapyrols,breakdown products of chlorophyll. AtMRP1 and AtPGP1,which have been implicated in auxin transport and arerelated to animal multidrug resistance (MDR) geneslocalize to the plasma membrane (Noh et al., 2001).

The prototype of these proteins contains two nucleotide-binding folds (NBFs) and two integral membrane domainswith multiple TMs, separated by each other. ABC-proteinsin Arabidopsis appear to be constructed in a modularmanner, as is the case for other organisms: the genomedisposes several half-molecules, containing a single ATP-binding domain fused to a membrane intrinsic domainwith multiple TMs. In addition, similar as in bacteria,soluble proteins with NBFs, but without TMs areencountered. Each NBF is composed of 200amino acidresidues containing a Walker A motif, a signaturesequence and a Walker B motif. More than 100 genesencode ABC-proteins in Arabidopsis, and about 80%

contain contiguous transmembrane spans. A completesummary of Arabidopsis genes belonging to this class oftransporters can be found in Sanchez-Fernandez et al.(2001).

Notable features of theArabidopsis ABC superfamily arethe presence of a large yeast-like PDR (pleiotropic drugresistance) subfamily, and the absence of genes encodingbona fide cystic fibrosis transmembrane conductanceregulator, sulfonylurea receptor, and heavy metaltolerance factor 1 (HMT1) homologs.

Passive transporters

Transporters that passively equilibrate the respectiveconcentrations across membranes are also sometimescalled carrier or permeases, without clear distinction in thetransport mechanism.A common feature of transporters is that the transport is

saturable at high substrate concentrations. This is easilyunderstood when transport of a substrate is followed step

by step (Fig.7). In the simplest scheme the substrates arebound to externally exposed binding sites, the transporterrearranges by a conformational change and releases thesubstrates in an ordered or unordered fashion on theinternal side. Binding of a substrate, translocation to theother membrane side and release reminds of enzymereaction kinetics, where a substrate is bound, modifiedand the new product is released. As long as intracellularconcentrations are low, transport and intracellular releaseare basically irreversible like an enzymatic reaction. Therate constant for binding and dissociation on the externalside, and the transport rate step are under theseconditions the moist relevant rate constants. The similarityin the underlying reaction is manifested in the fact thattransporters, like enzymes, show Michaelis-Mentenkinetics. The Michaelis constant Km in enzyme kinetics isdefined as the substrate concentration, where thecatalytic reaction is half-maximal. At high, saturatingsubstrate concentration the maximal reaction capacity v

max

is reached. Similarly, net uptake rate by a transporter ismaximal at J

maxand half-maximal at Michaelis constant K

m.

The uptake rate is resultant of influx and efflux. The half-maximal transport capacity of a transporter is one basicfunctional characteristic that can be easily determined(Fig. 8).

The kinetics can be easiest studied when the respectivetransporter is biochemically isolated or functionallyexpressed in heterologous systems. In the plant, wheregenerally many transporters contribute to net uptake orrelease, metabolism has to be considered additionally.Thus the measured kinetics in living organisms have to betaken with caution: enzymatic consumption or subsequentuptake into an internal compartment such as the largevacuole in plant cells may misrepresent the actual plasmamembrane uptake rates. For actual in vivo uptake kinetics

see for example Marschner (1993).The transport mechanism of some transporters has beenanalysed and described by kinetic rate models. In thesesimplified models the physical states of the transporterare represented and transitions between these states arethe rates that kinetically describe the transport (Fig. 7).Detailed model evaluations have been done for hexosetransport in AtSTP1(Boorer et al., 1994), sucrose transport

Figure 7. Schematic representations of reaction schemes forcotransporters. Kinetic (A) and physical (B) models to describe thetransport of a substrate (S) and a proton (H) to the carrier with itsbinding sites exposed to the external side (C

o) or to the internal

side (Ci)


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Figure 8. Dependence of transport activity of AAP5 on substrate(lysine) concentration A Lysine induced current versusconcentration in a linear Michaelis-Menten plot, B doublereciprocal plot. (unpublished)

by AtSUC1 and StSUT1 (Boorer et al., 1996; Zhou et al.,1998) and amino acid transport by AtAAP1 and AtAAP5(Boorer et al., 1996; Boorer and Fischer, 1998). Sincenone of the rate constants can be measured in isolation,the models must generally extract kinetic information fromsteady state situations. The simplest model for aH+/symporter involves 6 states and 12 rate constants,

which obviously leads to some flexibility of a single modelto describe the data (Fig. 7). Such flexibility is problematicwhen considering whether a specific model with definedrate constants exclusively describes the mechanism inone transporter. Although it may be arbitrary for thephysiology of the plant if a transporter is binding itssubstrates in an ordered fashion or not, it is veryinstructive to understand the principles of transport. Bothtransporters act as proton symporters with 1:1stoichiometry, and interestingly, StSUT1 may have a slipmodus, with sucrose transported without protons (Booreret al., 1996). Maltose is the only sugar transported withhigh efficiency (about half as good as sucrose), butmonosaccharides or higher order sugars are nottransported. Some cooperativity and voltage dependent

binding of uncharged sucrose is observed. The data havebeen interpreted in two slightly different ways: AtSUC1binds external protons and sucrose in non-orderedfashion, but on the internal side the sugar dissociates first(Zhou et al., 1997). StSUT1 binds protons before sucrose,both ligands are transported simultaneously across themembrane and sucrose dissociates first (Boorer et al.,

1996). Although both sucrose transporters may functiondifferently, the multiplicity of parameters in such modelsmay impair simple conclusions. Even a model with 6states may be only a minimal approximation of the realsituation.

An overview over metabolite transport systems inArabidopsis

The most advanced system regarding knowledge of thetransportome (i.e. the complete set of transporterproteins) of a eucaryote is the yeast Saccharomycescerevisae, which serves as a model system for many ofthe studies in Arabidopsis (van Belle and Andr, 2001).Completion of the genomic sequence of Arabidopsis hasallowed to generate membrane protein databasesproviding an overview over the potential number of

Arabidopsis transporters (Ward 2001; Schwacke et al.,submitted). According to the most recent analysis, the

Arabidopsis genome contains ~6475 proteins with at leastone membrane span or ~ 1976 proteins with at least fourmembrane spans (Schwacke et al., submitted). One mayassume that a large proportion of the proteins with atleast four membrane spans are involved in transport.Regarding functional assignment, transporters for the

Figure 9. Schematic view of a cell with intracellular compartments.Molecularly identified nutrient and metabolite transporters aredrawn in their respective membranes.


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most nutrients and many metabolites have been identifiedin Arabidopsis at the molecular level. Most transportersare members of multigene families, and only in rare casesall similar or homologous transporters have beeninvestigated in detail. The uptake transporters for nutrientsand metabolites face a general problem: usually cell hasto accumulate nutrients and metabolites against aconcentration gradient. On the other hand the cells haveto excrete waste products. If the cell would allowfacilitated diffusion, the quickly reached equilibrium wouldimpede further uptake. Only if the metaboliteconcentration within the cell decreases steadily, by activeassimilation or metabolism, the cell can act as a sink andwould maintain a steady influx.

Nutrient transporters

The uptake of nutrients in plants is of principle importancefor optimal growth and development. Besides theelements C, H and O, several more elements are essentialat high quantities, and thus plants have developed robustand efficient uptake systems for the macronutrients (N, K,Ca, Mg, P, S) from the rhizosphere. Of the micronutrientsCl, B, Fe, Mn, Zn, Cu, Mo, Ni, Na (Marschner, 1993) onlytrace amounts, generally about 100-10.000 fold less thanmacronutrients, are necessary for plant growth. However,secondary nutrient transporters are also involved in waterbalance, osmolyte equilibration and turgor generation. Thecell wall allows the plant cells to survive in a dilutemedium without bursting, which is in contrast to animalcells that strictly regulate the turgor in a small range byuptake and excretion of osmolytes.

Transporters for all macronutrients have been identified

inArabidopsis (Fig. 9). For e.g. potassium uptake, a cationcritically involved in turgor generation and establishmentof the membrane potential, many unrelated transporterswith diverse coupling mechanisms have been identified.An overview about transporter families involved inanorganic cation transport can be found in Maser et al.(2001), and will not be detailed here.

Several organic compounds and metabolites can betaken up by roots from the rhizosphere and may serve asnutrients. In the case of nitrogenous compounds, theprimary nitrogen sources in most soils are anorganicnitrate and ammonium. Ammonium and nitrate areacquired by use of high and low affinity transporters (NigelM. Crawford and Brian G. Forde; this book). Althoughammonium may be preferred due to the lower energetic

cost needed for assimilation (Gazzarrini et al., 1999),nitrate is often the main nitrogen source for plants. Afterconcentrative, electrogenic H+-coupled uptake into thesymplasm by epidermal and cortical cells, it may bestored in vacuoles, where concentrations up to severaltens of mM can occur. Nitrate is subsequently reduced ina two-step process to ammonium, or mobilized to the

xylem, and later reduced to ammonium in shoots. Bothlow and high affinity transporters have been molecularlyidentified. They belong to two structurally distinct families,the NTR1 (52 members, Tsay et al., 1993) and NTR2 (7members, Trueman et al., 1996) families, both with 12TMs. Interestingly, one barley homolog of the NTR1 familytransports histidine in addition to nitrate (Zhou et al.,1998), and one Arabidopsis homolog transportsdipeptides and histidine (Rentsch et al., 1995). This mayindicate that within a single protein family the requirementfor nitrogen leads to evolution of a capacity to transportseveral nitrogenous compounds. However, the details ofsubstrate recognition in such transporters remainobscure. High affinity acquisition of ammonium is due toAMT transporters (6 members), which facilitate NH4

+diffusion across membranes (Ninnemann et al., 1994;Ludewig et al., 2002). These proteins form multimers witheach subunit composed of 11 TMs. Nitrogen uptake andmetabolism is highly regulated and tightly linked to carbonmetabolism (Coruzzi and Bush, 2001, Corruzzi and Zhou,2001).

Other macronutrients, like sulfur and phosphorous maybe also taken up in form of organic molecules, but in mostsoils sulfur will be acquired by sulfate transporters, thatare secondary active and import SO4

2- by cotransport withthree protons, giving a net positive charge transport intothe cytoplasm. 12 members of that family with 12 putativeTMs are identified in the Arabidopsis genome (Takahashiet al., 2000).

Phosphate (PO43-) is, unlike nitrate and sulfate, not

reduced in plants during assimilation. It remains in theoxidized state and forms phosphate esters in manyorganic molecules. Many factors, including poor solubility,high sorption to soil particles, and competition forphosphate in the soil lead to low availability of phosphate

to the plant. Thus plants have evolved regulated highaffinity systems to cope with typical soil concentrations ofbelow one M. In addition, some plants modify therhizosphere to

Genes and Proteins for Solute Transport and Sensing

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