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Review TRENDS in Plant Science Vol.6 No.

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11 Yang, H. et al. (1999) Oryza sativa PSK gene development affecting the same processes as Science 286, 1697–1700
encodes a precursor phytosulfokine-α, a sulfated CLAVATA1. Development 121, 2057–2067 28 Takayama, S. et al. (2000) The pollen determinant
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genes encoding precursors for phytosulfokine, a 20 Trotochaud, A.E. et al. (1999) The CLAVATA1 kinase is inhibited by thioredoxins and activated
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13 Hanai, H. et al. (2000) Existence of a plant assembly into a signaling complex that includes 30 Saitoh, M. et al. (1998) Mammalian thioredoxin is
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a sulfated pentapeptide, stimulates the proliferation 22 Brand, U. et al. (2000) Dependence of stem cell 32 Kobe, B. and Deisenhofer, J. (1995) Proteins with
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Strategies to identify transport


systems in plants
Hélène Barbier-Brygoo, Frédéric Gaymard, Norbert Rolland and Jacques Joyard

Since the first molecular structures of plant transporters were discovered over molecular techniques and the use of heterologous
a decade ago, considerable advances have been made in the study of plant expression systems have allowed the identification of
membrane transport, but we still do not understand transport regulation. transporter genes and the characterization of the
The genes encoding the transport systems in the various cell membranes are encoded proteins, providing the first pictures of
still to be identified, as are the physiological roles of most transport systems. the molecular structures of plant transporters1,2. On
A wide variety of complementary strategies are now available to study the other hand, forward and reverse genetic approaches
transport systems in plants, including forward and reverse genetics, have enabled the physiological roles of transport
proteomics, and in silico exploitation of the huge amount of information systems in planta to be unraveled in a few cases.
contained in the completely known genomic sequence of Arabidopsis. A new era has begun with the sequencing of
Arabidopsis3. About 18% of the 25 498 predicted
Plant membrane transporters (pumps, carriers and proteins appear to contain two or more membrane
channels) participate in various functions including spans, whereas 5–10% represent transport proteins4.
mineral nutrition, carbon and nitrogen metabolism, Putative transport functions have been assigned to
cell signaling, osmoregulation, cell homeostasis, these proteins based on homology with already
storage and stress responses. Until recently, although identified counterparts in other organisms. However,
there was a huge amount of functional data analysing plant cells might have evolved unique transport
transporter activities in plant cells and their fine properties, and a major challenge remains to uncover
tuning by a variety of regulation mechanisms, plant-specific transport systems. Another challenge
information about the molecular bases of the arises from the highly compartmentalized
transport processes was scarce. The past decade has organization of plant cells and the expected
seen tremendous advances in the field of plant specialized transport equipment of each membrane
membrane transport. On the one hand, the advent of compartment. The aim of this article is to discuss

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578 Review TRENDS in Plant Science Vol.6 No.12 December 2001

various strategies that might help to identify and mutant by the expression of an heterologous
characterize genes encoding plant transport systems, transporter18–20. Thus, functional complementation
and to try to make sense out of the emerging of a strain defective in a specific transport pathway
complexity of transport regulatory networks. does not necessarily demonstrate that the cloned
gene encodes a plant transport system.
Plant transporters in heterologous systems: how far Xenopus oocytes and other animal cells such as
can we go? insect or COS cells have mainly been used for
A major breakthrough in our knowledge of studying the functional properties of cloned channels
membrane transport in plants occurred with the use and transporters16. Xenopus oocytes were also used
of heterologous expression systems for cloning in cloning strategies enabling the identification of
membrane transport genes and/or characterizing the several animal channels and transporters21.
functional properties of the corresponding proteins. Surprisingly, in spite of a report describing the
Several cloning strategies have been described to expression of a maize K+ channel after the injection
isolate membrane proteins based on the detection of of cRNA in oocytes22, this strategy has not been used
proteins expressed at the surface of host cells. successfully to isolate plant cDNAs.
Immunoselection of membrane proteins has been Yeast allowed the expression cloning of many plant
performed in both animal cells (COS cells from transport systems, mostly residing in the plasma
green monkey kidney)5,6 and E. coli7. Other systems membrane of their native cells, whereas both plasma
have been developed, both in COS cells and in membrane and tonoplast proteins could be
yeast, based on the detection of targeting of a functionally expressed in Xenopus oocytes. A great
peptide of known structure or function to the limitation of both these systems is that few (if any)
membrane: the signal sequence trap8,9. However, membrane proteins from organelles (chloroplast and
these topology-based screening strategies seem to be mitochondria) came out of these functional screens.
more efficient for secreted and type-I membrane The main reason might be the presence of a transit
proteins, with the multiple-transmembrane-segment peptide preventing the protein from reaching the
proteins – transporters belong mainly to this last plasma membrane of yeast cells or oocytes, where its
type – being rarely selected. activity could be detected. In some instances, once a
Although E. coli is the easiest expression system gene has been identified, no transport activity
for protein production, it has been little used in the corresponding to the expressed protein can be
membrane protein field because incorrect folding and detected after expression in heterologous systems. In
targeting of hydrophobic eukaryotic proteins is other cases, such as stomatal guard cells, pronounced
common. Many overexpressed membrane proteins differences have been observed between the activity
seem to be toxic or expressed as unfolded inactive of the native K+ channels and that of cloned
proteins. Only a few reports describe the functional α-subunits expressed in Xenopus oocytes16. Several
expression of a membrane protein10–12 and E. coli has hypotheses can account for these negative results.
mainly been used to determine the membrane The plant protein might be incorrectly matured or be
topology of proteins. targeted to a membrane compartment that is
Among the heterologous systems used, the different from the plasma membrane. Alternatively,
unicellular yeast Saccharomyces cerevisiae has the transport system might require plant-specific
Hélène Barbier-Brygoo proved to be the most powerful tool, mainly because protein partners that are not available in the
Institut des Sciences du many strains defective in specific transport activities heterologous system; for example, if the functional
Végétal, UPR 2355, CNRS, are available. Most of our current knowledge about transport unit results from the heteromeric assembly
91198 Gif-sur-Yvette
Cedex, France.
the molecular basis of plant transport has been of different subunits23. In addition, cell-type-specific
e-mail: gained using functional complementation strategies post-translational modifications and/or the presence
brygoo@isv.cnrs-gif.fr on yeast mutants or strains with modified metabolic of cytosolic compounds might contribute to transport
Frédéric Gaymard pathways. In the early 1990s, an invertase-deficient activity or regulation. In this context, even when a
Laboratoire de Biochimie mutant unable to hydrolyse extracellular sucrose was transport activity can be detected upon heterologous
et Physiologie engineered to express an intracellular sucrose expression, caution must be exercised in directly
Moléculaire des Plantes,
UMR 5004 (CNRS/INRA/
synthase gene, and several sucrose transporter transposing the data to speculate about the
ENSA-M/Université cDNAs were cloned using this strain. Later, yeast transporter functions in its native cell.
Montpellier 2), INRA, strains with defective K+-uptake systems were
34060 Montpellier Cedex 1,
complemented by Arabidopsis cDNA libraries, Classical (forward) genetics: which screens?
France.
which enabled two K+ channel cDNAs to be isolated. The isolation of plant mutants altered in specific
Norbert Rolland After these first successes, yeast mutants in various transport pathways is potentially a powerful way to
Jacques Joyard
Laboratoire de
transport systems were used to clone the identify genes involved in ion or metabolite transport.
Physiologie Cellulaire corresponding plant genes using this effective Screens might include the search for mutants altered
Végétale, UMR 5019 strategy13–17. However, expression of a foreign protein in their growth response to exogenous ions24, over- or
(CNRS/CEA/Université
in yeast can interfere with its cellular physiology and underaccumulating specific ions25,26, or mutants
Joseph Fourier),
CEA-Grenoble, 38054 the functional complementation might not be due to a altered in their sensitivity to compounds inhibiting
Grenoble Cedex 9, France. replacement of the deficient transport capacity of the transport protein activities27,28. Apart from isolating

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Review TRENDS in Plant Science Vol.6 No.12 December 2001 579

many mutants and physiologically characterizing membrane and solubilized with either organic
them, genetic approaches for identifying transport solvents35 or nonionic or zwitterionic detergents38
protein genes have rarely been successful. Only two before electrophoresis. However, of the Arabidopsis
cases have been reported to date, which led to the plasma membrane proteins that have been
identification of a nitrate uptake system29 and of a separated by 2D PAGE after solubilization with
protein homologous to the Na+–H+ antiporters found various detergents (e.g. C8Ø, ASB14), only a few are
in fungi and animals30. Surprisingly, there are more transport proteins: an aquaporin (PIP, monomer and
examples in the literature of mutants being selected dimer) and the H+-ATPase, which have six and ten
on the basis of altered developmental processes that, transmembrane domains, respectively, and (from
when the mutated genes were cloned, turned out to roots) a high-affinity nitrate transporter (NRT2)
involve transport systems31–34. For instance, the dnd1 with 12 transmembrane domains38.
(defense, no death 1) mutant fails to produce a Using chloroplast envelope membranes as a
hypersensitive response and exhibits constitutive model, the solubility of hydrophobic proteins in
systemic resistance and elevated levels of salicylic organic solutions has been used to extract them
acid. DND1 was shown to be a cyclic-nucleotide-gated from purified membrane fractions35 (Fig. 1).
channel of previously unknown in planta function32. Polypeptides were separated by one-dimensional
These examples show that, in some cases, genetic SDS-PAGE, analyzed by tandem mass
strategies based on functional screens related to spectrometry microsequencing and characterized
transport activities can lead to the identification of by comparison with protein sequences deduced
key steps in transport pathways. However, such a from genomic sequences present in the databases
strategy requires a huge amount of work and has led (see below). Most of the envelope polypeptides
to the identification of few transporters. In addition, soluble in chloroform–methanol mixture had
although it is based on transport processes, such 1–13 putative transmembrane α-helices35. The
screening can lead to the isolation of mutations with propensity of hydrophobic proteins to partition into
pleiotropic effects or to the identification of genes a chloroform–methanol mixture was directly
involved in the regulation of ion homeostasis rather correlated to the Res:TM ratio (the ratio of the
than in transport steps. In other cases, transporter number of amino acid residues to the number of
genes have been isolated by accident because there putative transmembrane regions), with a cut-off
was no way to predict that the selected developmental value of ~100. For instance, two important
phenotype would be associated with a mutation in a envelope membrane transporters were identified
transporter gene. These two types of statements do in the chloroform–methanol-soluble fraction:
not argue in favor of the use of forward genetics as the the malate–oxoglutarate translocator
most appropriate approach to target transporter (12–13 transmembrane α-helices, Res:TM ratio of 38)
genes specifically. (Fig. 1) and the phosphate–triose-phosphate
translocator (6–7 transmembrane α-helices,
Identifying plant transporters using proteomics Res:TM ratio of 49). In addition, this fraction
The use of proteomics as a method to identify all contains a series of putative transporters with
cellular proteins is currently in a rapid state of molecular masses ranging from 10 kDa to 100 kDa
development, and its use to identify transport and contains one or more putative membrane-
proteins is just emerging2,35,36. Two main features of spanning domains. Such proteins do not
membrane transporters make them difficult to correspond to any previously identified chloroplast
identify using proteomics. First, transporters are envelope transporters but have homologies to
often minor components in cell membranes. Their several prokaryotic transporters35. Thus,
low abundance can impede their identification and chloroform–methanol selective extraction is a
analysis in plant tissues even by sensitive techniques versatile method to recover minor, highly
such as mass spectrometry2,35. A solution to this hydrophobic proteins from purified membrane
problem is to analyze subcellular proteomes starting systems35,39. However, the proteins that might
from isolated membranes and vesicles. Second, most correspond to transport systems must still be
transporters contain a series of 15–25 nonpolar identified from among these hydrophobic proteins.
amino acids constituting transmembrane α-helices This question should be first addressed in silico.
buried in the hydrocarbon core of the membrane.
Such proteins are therefore highly hydrophobic and Bioinformatics: linking genomics and functional analysis
are under-represented in two-dimensional (2D) Large-scale genome sequencing efforts, such as the
patterns obtained by polyacrylamide gel Arabidopsis Genome Project3 (Box 1), and the resulting
electrophoresis of membranes37. This under- massive amount of data provide the basis for computer-
representation is due (at least in part) to the aided analyses to predict all the open-reading-frames
currently available proteomic technology, which to be encoded by these genomes. General information
favors the specific loss of the most hydrophobic is mostly concentrated on web sites dedicated to
membrane proteins. This is true for all membranes genome annotations and displaying information on
unless intrinsic proteins were extracted from the predicted proteins from several plant species. One way

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580 Review TRENDS in Plant Science Vol.6 No.12 December 2001

Step 1: Purification of chloroplast envelope membranes

Step 2: Extraction of hydrophobic proteins by chloroform and methanol

Step 3: Proteomic analyses

(a) (b)
I S
kDa MASMALSLTS SPTYSLSFRS LPSLKPLSKS QPSISLPSLR SNASKSPSLS HKHFLSPPSL
LLPHKLKPIS ASSPTNPPPP PAPVPSPAPV SAPAQVQPWQ GASIKPLLAS ILTGVIIWFI
84.0 PTPEGVSRNA WQLLAIFLST IVGIITQPLP LGAVALMGLG ASVLTKTLTF SAAFSAFGDP
IPWLIALAFF FARGFIKTGL GNRIAYQFVK LFGSSSLGLG YSLVFSEALL APAIPSVSAR
AGGIFLPLVK SLCIACGSNV GDGTERKLGA WLMLTCFQTS VISSSMFLTA MAANPLSATL
53.0
TFNTIGKAIG WMDWAKAAFV PGLVSLIVVP LLLYVVYPPE IKSSPDAPRL AKEKLDKMGP
IE45 MTKNESIMAV TLLLTVGLWV FGGKLGVDAV TAAILGLSVL LITGVVTWKE CLAESVAWDT
LTWFAALIAM AGYLNKYGLI TWFSENVVKV VGGLGLSWQM SFGVLVLLYF YSHYFFASGA
34.9 IE37 AHIGAMFTAF LSVASALGTP PFLAAIVLSF LSNLMGGLTH YGIGSAPVFY GANYVPLPQW
IE30 WGYGFLISIV NLIIWLGVGG LWWKAIGLW
28.7

TMpred output for IE45


20.5 (d) 3000
IE18
IE16 2000
1000
(c) MALDI-TOF 0

a.i. –1000
–2000
X
10000 –3000
–4000
X
5000 –5000
X 0 100 200 300 400 500 600

900 1100 1300 1500 1700 1900 2100 m/z TRENDS in Plant Science

Fig. 1. A proteomic strategy to identify membrane transporters. Step 1 (MS/MS) for microsequencing (b) or by matrix-assisted laser desorption
is the purification of membranes from plant tissues or cells. The ionization time-of-flight mass spectrometry (MALDI-TOF) to obtain peptide
envelope membranes must be properly characterized and the level of mass fingerprints (c). The poor resolution of one-dimensional PAGE is
their purity thoroughly determined35. In Step 2, the hydrophobic proteins compensated for by the high sensitivity and remarkable ability of MS/MS
are extracted from envelope membranes with a mixture of chloroform and to characterize small quantities of proteins, even in complex mixtures.
methanol35. Envelope polypeptides are then separated by one-dimensional (b) Proteins can be identified by comparison of peptide sequence tags
SDS-PAGE (a), instead of two-dimensional PAGE, to eliminate the risk of loss with protein sequences deduced from genomic sequences present in the
of hydrophobic proteins during electrophoresis. In addition, SDS is efficient databases. The protein sequence shown is that of the malate–oxoglutarate
at solubilizing highly hydrophobic proteins. Furthermore, most chloroplast translocator, a major chloroplast envelope transporter of 45 kDa (Ref. 40).
envelope transporters identified to date are highly basic proteins and are Amino acids in blue correspond to the chloroplast targeting sequence; those
only poorly resolved in the best cases, when using commercially available in red correspond to the sequences obtained by MS/MS. Underlined amino
immobilized pH gradients. A small proportion (5–10%) of the total envelope acids correspond to the 12–13 putative transmembrane α-helices. (c) The
polypeptides is soluble (S) in chloroform–methanol, whereas most circles above the peptide mass fingerprint represent masses of some tryptic
membrane polypeptides remain in the chloroform–methanol-insoluble fragments of the envelope malate–oxoglutarate translocator, whereas
phase (I). Polypeptides separated by SDS-PAGE are digested by trypsin (in- the X represents the masses of some trypsin peptides. (d) Hydropathy
gel digestion) and the peptides then analyzed by tandem mass spectrometry profile of the chloroplast envelope malate–oxoglutarate translocator.

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Review TRENDS in Plant Science Vol.6 No.12 December 2001 581

been created (Box 1). The ‘manually assigned


Box 1. The global bioinformatic analysis – from the Arabidopsis thaliana
functional catalog’ of the MATDB site provides
genome sequence to the prediction of plant membrane transporter families
tables and catalogs of functional categories,
Arabidopsis thaliana genome sequencing: The Arabidopsis Genome including transporter families, for the hypothetical
Initiative (AGI) http://www.arabidopsis.org/ Arabidopsis proteins. The AMPL is a collection of
Cold Spring Harbor Sequencing Consortium (CSHSC) almost 4600 predicted polytopic Arabidopsis
http://nucleus.cshl.org/protarab/ membrane protein sequences (containing at least
European Scientists Sequencing Arabidopsis (ESSA) two predicted membrane-spanning domains) that
http://mips.gsf.de/proj/thal/proj/proj_overview.html have been clustered into families based on sequence
Genoscope-EU Consortium homology4, a subsection being dedicated to
http://www.genoscope.cns.fr/externe/English/Projets/Projet_S/S.html Arabidopsis transporter families (‘Arabidopsis
Kazusa DNA Research Institute http://www.kazusa.or.jp/kaos/ transporter view’). The ‘Genomic comparisons of
SPP Consortium http://sequence-www.stanford.edu/ara/SPP.html membrane transport systems’ site contains the
The Institute for Genomic Research (TIGR) http://www.tigr.org/ distribution of known and putative polytopic
membrane transport proteins for all organisms for
Plant gene predictions which completely sequenced genomes are available.
NetPlantGene (splice site prediction) Transport systems for each organism were
http://www.cbs.dtu.dk/services/NetPGene/ classified according to putative membrane topology,
GeneMark (coding probability of single exons) protein family, bioenergetics and substrate
http://genemark.biology.gatech.edu/GeneMark/gm_info.html specificity. Complete lists of the transporters from
XGrail (coding probability of single exons) http://compbio.ornl.gov/Grail-1.3/ each organism are provided. The ‘Transport
GENSCAN (gene modeling) proteins in Arabidopsis’ page displays graphical
http://genome.dkfz-heidelberg.de/cgi-bin/GENSCAN/genscan.cgi data about almost 900 classified Arabidopsis
GENEFINDER (gene modeling) transporters. Recently, huge efforts have been
http://mips.gsf.de/proj/thal/proj/genefinder.html made to analyze specific subfamilies of plant
TRNAscan-SE (detection of transfer RNA genes) transporters. Inventories and phylogenic
http://www.genetics.wustl.edu/eddy/tRNAscan-SE/ relationships within subfamilies of Arabidopsis
cation transporters41, ABC proteins42, P-type
Plant genome annotation (general) pumps43, MIPs (Ref. 44) and maize aquaporins45
MATDB (MIPS Arabidopsis thaliana database) data of the AGI compiled, have recently been published, and corresponding
analyzed, annotated and stored http://mips.gsf.de/proj/thal/db/index.html Web sites have been created (Box 1).
Center for Medicago Genomics Research The first step after identifying a new protein
http://www.noble.org/medicago/index.htm sequence is the search for homology between the
ZmDB (Zea mays DataBase) http://www.zmdb.iastate.edu/ sequence and those from various databases (Box 2;
CSHL (maize genome analysis) http://nucleus.cshl.org/maizegenome/ Fig. 2). This is often the simplest way to obtain
CSHL (rice genome analysis) http://nucleus.cshl.org/riceweb/ functional information when it is available. However,
TIGR (The Institute for Genomic Research) (includes gene index pages for functional annotation of a sequence through
12 plant species) http://www.tigr.org/tdb/tgi.shtml comparisons encounters a major limitation: almost
TOGA (TIGR Orthologous Gene Alignment database) (Includes orthologs half of the proteins predicted in Arabidopsis are
from Arabidopsis, rice, tomato, potato, Medicago, soybean, maize, unrelated to any protein with a known function46, and
wheat) http://www.tigr.org/tdb/toga/toga.shtml functional information is still lacking for 60% of the
Arabidopsis membrane protein sequences4.
Functional libraries including plant transport systems When the sequence of an unknown protein is too
MATDB tables (functional categories) distantly related to any protein of known structure
http://mips.gsf.de/proj/thal/db/tables/tables_func_frame.html to detect its resemblance by overall sequence
AMPL (Arabidopsis Membrane Protein Library) alignment, relationships can be revealed by the
http://www.cbs.umn.edu/arabidopsis/ occurrence of a particular cluster of residue types in
Genomic comparisons of membrane transport systems its sequence (Box 2). The use of protein sequence
http://www-biology.ucsd.edu/~ipaulsen/transport/ patterns, profiles, motifs, signatures or fingerprints
PlantsT (functional genomics of plant transporters) http://plantst.sdsc.edu/ to determine the function of proteins offers an
P-type ATPase database http://biobase.dk/%7Eaxe/Patbase.html alternative to more classical alignment programs47.
Gene families encoding aquaporins These databases help to determine which known
http://mbclserver.rutgers.edu/CPGN/AquaporinWeb/Aquaporin.group.html family of protein (if any) a new sequence belongs to,
or which known domain(s) it contains. Many of the
most powerful sequence analysis methods are now
of making sense of the huge amount of data that is based on principles of probabilistic modeling.
being generated by high-throughput DNA technology Examples of such methods include the use of
is to get information on the predicted proteins. probabilistically derived score matrices to determine
Several web sites displaying information on the significance of sequence alignments, the use of
predicted plant transporter families have recently hidden Markov models as the basis for profile

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582 Review TRENDS in Plant Science Vol.6 No.12 December 2001

Efficient tools are also available for other


Box 2. The reverse bioinformatic analysis – from a unique sequence to
analyses. The function of a protein is tightly
the prediction of a plant membrane transporter
correlated with its subcellular localization, which is
Homology search tools linked to its physicochemical nature. There are tools
BLAST (NCBI Web Site) queries performed on non redundant or species- to predict transmembrane domains in protein
specific databases (includes Arabidopsis, rice, maize) sequences (Box 2) or protein sorting signals, signal
http://www.ncbi.nlm.nih.gov/BLAST/ peptide or cleavage sites (Box 2). Some methods are
TAIR (The Arabidopsis Information Resource) http://www.arabidopsis.org/ based on the hydrophobicity plot of the analyzed
protein (e.g. physicochemical profiles) or on
Protein families predictions comparisons to a transmembrane database
PFAM (database of protein domain families) http://www.sanger.ac.uk/Pfam/ (e.g. TMpred), whereas others predict the location
PRODOM (protein domain database) and topology of transmembrane helices from multiple
http://protein.toulouse.inra.fr/prodom.html sequence alignments (e.g. TMAP, PHDhtm). A hidden
BLOCKS (blocks database) http://www.blocks.fhcrc.org/about_blocks.html Markov model was recently developed to predict the
PRINTS (protein motif fingerprint database) topology of helical transmembrane proteins
http://www.bioinf.man.ac.uk/dbbrowser/PRINTS/ (HMMTOP). This method is based on the hypothesis
PROSITE (dictionary of protein sites and patterns) that the localization of the transmembrane
http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html segments and their topology are determined by the
difference in the amino acid distributions in various
Hidden Markov models for profile predictions structural parts of these proteins rather than by
MEME (multiple EM for motif elicitation for discovering motifs in a group specific amino acid compositions of these parts.
of related protein sequences) http://meme.sdsc.edu/ However, this apparently obvious approach is not
HMMER (profile hidden Markov Models for biological sequence analysis: always satisfactory, and information obtained by
provides statistical models of the primary structure consensus of a predicting sorting signals is not reliable. Gene
protein family) http://hmmer.wustl.edu/ assignments are often unreliable in their 5′ regions49,
the analyses of which are usually the basis of the
Prediction of helical transmembrane proteins prediction of protein sorting signals. Other
TMpred (prediction of membrane-spanning regions and orientation based limitations might exist: for instance, porins present
on the statistical analysis of a database of naturally occurring in outer membranes from plastids and
transmembrane proteins) mitochondria50 do not contain any cleavable (and
http://www.ch.embnet.org/software/TMPRED_form.html therefore predictable) N-terminal transit sequence
PSORT Prediction (recognition of most probable transmembrane segments or any transmembrane α-helices.
from the average hydrophobicity value of 17-residue segments)
http://psort.nibb.ac.jp/helpwww.html#src Reverse genetics: a necessary gateway to integrated
Physico-chemical profiles (protein sequence analysis for displaying the transport function
hydropathic character of a protein) http://pbil.univ-lyon1.fr/ In spite of the growing amount of information on
TMAP (prediction from multiple sequence alignment) protein sequences and functional characterization,
http://www.mbb.ki.se/tmap/ the fundamental question about the physiological role
SOSUI (classification and secondary structure prediction of membrane in planta remains to be clarified for most transport
proteins) http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html systems. The isolation and characterization of loss-of-
HMMTOP (hidden Markov model for topology prediction of helical function mutants is a powerful way to assign a
transmembrane proteins) http://www.enzim.hu/hmmtop/index.html biological function to a known gene. In many model
PHD (suite of programs predicting 1D structure from multiple sequence organisms (yeast, bacteria, mice), homologous
alignments. PHDhtm predicts the location and topology of recombination can be used efficiently to target
transmembrane helices) http://dodo.cpmc.columbia.edu/predictprotein/ mutations to specific genes. In plants, homologous
recombination has been described but appears to be
Subcellular localization (compatible with plant input sequences) difficult to implement and is not feasible on a large
ChloroP v1.1 (chloroplast transit peptide prediction) scale, except for the moss Physcomitrella patens51.
http://www.cbs.dtu.dk/services/ChloroP/ An alternative, highly efficient procedure for
TargetP v1.01 (prediction of subcellular location) isolating mutants in identified genes is insertional
http://www.cbs.dtu.dk/services/TargetP/ mutagenesis. The improvement of plant
Predotar (prediction of putative mitochondrial and plastid targeting transformation techniques52 has led to the formation
sequences) http://www.inra.fr/Internet/Produits/Predotar/ of large collections of plants mutagenized by T-DNA,
PSORT Prediction (Prediction of subcellular location) insertion elements or transposons53,54. Several
http://psort.nibb.ac.jp/helpwww.html#src collections of plants or DNA pools and databases of
flanking sequence tags (databases of systematic
sequencing of insertion sites) can be screened for the
searches to identify distant members of sequence identification of lines harboring insertions within a
families (Box 2) and the inference of phylogenetic gene of interest. Moreover, recent reports of RNA-
trees using maximum likelihood approaches48. mediated interference have opened new prospects for

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Review TRENDS in Plant Science Vol.6 No.12 December 2001 583

also might arise from our inability to detect weak,


Membrane Membrane subtle physiological changes54.
compartments compartments
In the field of transport proteins, the physiological
Antibodies Genomic sequences Hydrophobic proteins roles of several proteins have been assessed or
ESTs partially resolved by reverse genetics. Ten mutants
Mass spectrometry disrupted in transporter genes have been reported
SST in
COS cells (Table 1). In some cases, the isolation and
or yeast Search for characterization of mutants brought genetic
homologs in Peptide sequences
databases evidence of the expected physiological function of the
corresponding genes57,59–61. Some studies revealed
Membrane Homologs in unexpected roles of the transport systems58,62 and
protein Cyanobacteria,
Chlamydomonas,
others depicted pleiotropic phenotypes63 or did not
libraries cDNAs GFP fusions
cDNA pools Physcomitrella allow clear assessment of the role of the gene in the
or libraries
Heterologous
databases plant64 owing to the lack of functional characterization
Promoter–reporter
expression fusions (e.g. GUS) Homologous of the corresponding transport system. These
(yeast, oocytes, Gene recombination examples show that knowledge of gene expression
insect cells,
COS cells) patterns associated with functional characterization
Cellular and
subcellular Knock-out have provided clues that orientated the phenotypic
localization mutants characterization of the mutants, leading to the
Functional Loss of identification of the physiological role of the transport
characterization function
mutants
system. The subcellular localization of a transporter
Phenotype
is also a key piece of functional information, although
analyses
Phenotype it is rarely achieved in current studies65. Proteomics
Transport mutants analyses of specific membrane compartments as well as the
use of green fluorescent protein–transporter fusions
In planta function to label the target membrane appear to be the tools
TRENDS in Plant Science of choice to get such information (Fig. 2). Thus,
reverse genetics appears to be a powerful tool for
Fig. 2. Overview of the different strategies presently developed to identify transport systems in assigning a function to identified genes only when it
plants. Candidate sequences are provided by mining genomic and EST databases and membrane is combined with physiological approaches, functional
protein libraries, or by proteomic analyses of purified membranes (yellow boxes). Functional
information on putative transporters are expected to be obtained from expression in heterologous
studies, gene expression analyses at the organ tissue
systems and studies of cellular and subcellular localization (grey boxes), together with and cell levels, and identification of the target
characterization of loss-of-function mutants. The use of other model organisms where targeted gene membrane compartment.
replacement is possible can also be envisaged (blue boxes). The core strategy (red arrows), from
gene sequence to transporter function in planta, is fed by a combination of molecular, genetic and
physiological approaches (black and blue arrows).
Conclusions
The toolbox for functional genomics of plant membrane
sequence-specific inhibition of gene function or of all transporters comprises a set of approaches depicted in
the members of a multigene family55. Most collections Fig. 2. The identification of transporter genes might
and databases were developed in Arabidopsis but, be achieved in various complementary ways, based on
for a few years, considerable effort has gone into sequence homology, functional properties or membrane
building genomic (expressed sequence tags and localization. A combination of molecular, genetic and
bacterial artificial chromosomes) and genetic physiological approaches is then required to discover
(insertional mutants) resources in other plants such the roles of these transporters in the plant. Analyses
as maize56, rice and Medicago truncatula (Box 1). of the spatial and temporal expression patterns
Although these resources have been available to (regulation at the transcriptional, post-transcriptional
the scientific community for several years, only a few and translational levels, and localization at the organ,
mutants have been reported in the literature to date. tissue and subcellular levels) can provide information
In many cases, mutants do not display any clear about cellular and developmental functions.
phenotype when grown in standard conditions and Expression in heterologous systems is expected to
disruptions do not lead to dramatic changes in provide functional information about the candidate
growth or morphology. Several hypotheses have proteins, especially in terms of transport activity,
been proposed to explain the lack of clear phenotypes. permeability and substrate selectivity. It is also
First, it might come from partial or total functional expected that the characterization of loss-of-function
redundancy. In this case, phenotypes would only be mutants should permit the identification of
observed in double or triple mutants disrupted in informative phenotypic traits that should provide a
redundant genes. Second, compensation processes direct clue to gene function. However, as already
might occur, as observed for the skor-1 (Ref. 57) and discussed, once the knockout mutant has been isolated,
clca-1 (Ref. 58) mutants, in which decreased levels of the search for a phenotype often turns into a desperate
K+ and NO3− were compensated for by increases in hunt54. Similarly, other model organisms in which
Ca2+ and organic acid content, respectively. Third, it sequence data are available and targeted gene

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584 Review TRENDS in Plant Science Vol.6 No.12 December 2001

Table 1. Characteristics of Arabidopsis insertion mutants within channel and transporter genes identified by reverse geneticsa
Gene Transport Gene expression patterns Mutant Phenotypic traits Proposed physiological function(s) Refs
system growth
AKT1 K+ inward Root apex Reduced Roots lacking inward K+ currents, Root K+ uptake from the soil solution 59
channel at low K+ reduced K+ uptake
SKOR K+ outward Root stelar tissues down- Normal Lower shoot K+ content, lower K+ release into the xylem sap 57
channel regulated by ABA K+ concentration in xylem sap towards the shoot
KAT1 K+ inward Guard cells Normal Altered K+ currents, normal Contribution to stomatal function? 62
channel stomatal opening
CNGC1 Cyclic-
nucleotide-gated ND Normal Pb2+ tolerance, lower Pb2+ Pb2+ entry (metal or Ca2+ transport?) 64
channel accumulation
AtCLC-a Anion channel Aerial parts and roots Normal Lower shoot and root NO3− content General control of nitrate content 58
up-regulated by nitrate hypersensitivity to chlorate
AtNRT2 Nitrate transporter Roots repressed by Not known Impaired in high affinity NO3− High affinity NO3− uptake by roots 60
nitrate influx
AtSUC2 Sucrose Companion cells in Altered Stunted growth, retarded Apoplast phloem loading of sucrose 61
transporter phloem tissues development, sterility
AtNRAMP3 Metal transporter Aerial parts and roots Normal Moderate Cd2+ resistance Cd2+ transport, regulation of iron 68
up-regulated in roots transport
by Fe starvation
AtMRP5 ABC transporter Vascular tissues, Altered Decreased root growth, increased Auxin conjugate transport? Ion 63
epidermis (guard cells) lateral root formation channel regulation?
AHA4 H+-ATPase Root endodermis, Reduced Resistance of stomates to Control of ion homeostasis and 69
flowers glibenclamide. Increase in the nutrient transport
Na to K ratio upon salt stress
aAbbreviations: ABA, abscisic acid; ND, not determined.

replacement is possible, such as cyanobacteria, the challenges for future research. The first will be to
green alga Chlamydomonas reinhardtii (only for elucidate the specific roles of individual members of
chloroplast-encoded genes) or the moss P. patens, transporter gene families, in specific cells and/or in
should be considered. The search for homologs of plant response to developmental and environmental
transporter genes in these species to generate signals. Little is yet known about the mechanisms
knockout mutants and analyze their phenotype might controlling transporter activity at the post-
be of great help to direct phenotype studies on the translational level, and the identification of
corresponding plant mutants. Global methods such as interacting proteins will represent a first step
large-scale expression profiling of Arabidopsis genes towards an integrated view of transport processes.
and ionic and metabolic profiling can also be used to Finally, further knowledge of the tertiary structure
compare wild-type and mutant plants in a wide range of transporters and their structure–function
of developmental and environmental conditions, and relationships will require efforts dedicated to the
are expected to reveal unforeseen links between the crystallization of these membrane proteins.
disrupted gene and the activation or repression of Plant genome projects and functional genomic
various pathways and networks. studies have extended far beyond Arabidopsis and
As emphasized by Sarah Assmann2, until recently, now embrace a diverse collection of plant species
it was a significant accomplishment simply to clone (Box 1). In this broader perspective, precisely mapping
an ion transport gene, but we are now entering a new transporters in Arabidopsis within specific subcellular
era in which we have to make sense out of hundreds of compartments and defining the associated cellular
sequences encoding putative transporters. This is and in planta functions should provide relevant bases
accompanied by a necessary shift to high-throughput for the development of effective strategies to
methods for protein identification, gene expression manipulate important physiological processes for
studies, measurement of transport activities and agricultural purposes, such as plant nutrition or plant
mutant screening, which also opens exciting adaptation to abiotic or biotic stresses66,67.
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