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11 Yang, H. et al. (1999) Oryza sativa PSK gene
encodes a precursor phytosulfokine-α, a sulfated
peptide factor found in plants. Proc. Natl. Acad.
Sci. U. S. A. 96, 13560–13565
12 Yang, H. et al. (2001) Diversity of Arabidopsis
genes encoding precursors for phytosulfokine, a
peptide growth factor. Plant Physiol. 127, 842–851
13 Hanai, H. et al. (2000) Existence of a plant
tyrosylprotein sulfotransferase: novel plant
enzyme catalyzing tyrosine O-sulfation of
preprophytosulfokine variants in vitro. FEBS
Lett. 470, 97–101
14 Matsubayashi, Y. et al. (1997) Phytosulfokine-α,
a sulfated pentapeptide, stimulates the proliferation
of rice cells by means of specific high- and
low-affinity binding sites. Proc. Natl. Acad. Sci.
U. S. A. 94, 13357–13362
15 Matsubayashi, Y. and Sakagami, Y. (1999)
Characterization of specific binding sites for a
mitogenic sulfated peptide, phytosulfokine-α, in
the plasma-membrane fraction derived from
Oryza sativa L. Eur. J. Biochem. 262, 666–671
16 Matsubayashi, Y. and Sakagami, Y. (2000) 120-
and 160-kDa receptors for endogenous mitogenic
peptide, phytosulfokine-α, in rice plasma
membranes. J. Biol. Chem. 275, 15520–15525
17 Tang, K. et al. (2000) Plant regeneration from
protoplasts of a commercial Asian long-grain
javanica rice. Plant Cell Tissue Organ Cult. 60, 79–82
18 Clark, S.E. et al. (1995) CLAVATA3 is a specific
regulator of shoot and floral meristem
development affecting the same processes as
CLAVATA1. Development 121, 2057–2067
19 Clark, S.E. et al. (1997) The CLAVATA1 gene encodes
a putative receptor kinase that controls shoot and
floral meristem size in Arabidopsis. Cell 89, 575–585
20 Trotochaud, A.E. et al. (1999) The CLAVATA1
receptor-like kinase requires CLAVATA3 for its
assembly into a signaling complex that includes
KAPP and a Rho-related protein. Plant Cell
11, 393–406
21 Fletcher, J.C. et al. (1999) Signaling of cell fate
decisions by CLAVATA3 in Arabidopsis shoot
meristems. Science 283, 1911–1914
22 Brand, U. et al. (2000) Dependence of stem cell
fate in Arabidopsis on a feedback loop regulated
by CLV3 activity. Science 289, 617–619
23 Trotochaud, A.E. et al. (2000) CLAVATA3,
a multimeric ligand for the CLAVATA1
receptor-kinase. Science 289, 613–617
24 Cock, J.M. and McCormick, S. (2001) A large
family of genes that share homology with
CLAVATA3. Plant Physiol. 126, 939–942
25 Stein, J.C. et al. (1991) Molecular cloning of a
putative receptor protein kinase gene encoded at
the self-incompatibility locus of Brassica oleracea.
Proc. Natl. Acad. Sci. U. S. A. 88, 8816–8820
26 Takasaki, T. et al. (2000) The S receptor kinase
determines self-incompatibility in Brassica
stigma. Nature 403, 913–916
27 Schopfer, C.R. et al. (1999) The male
determinant of self-incompatibility in Brassica.
Science 286, 1697–1700
28 Takayama, S. et al. (2000) The pollen determinant
of self-incompatibility in Brassica campestris.
Proc. Natl. Acad. Sci. U. S. A. 97, 1920–1925
29 Cabrillac, D. et al. (2001) The S-locus receptor
kinase is inhibited by thioredoxins and activated
by pollen coat proteins. Nature 410, 220–223
30 Saitoh, M. et al. (1998) Mammalian thioredoxin is
a direct inhibitor of apoptosis signal-regulating
kinase (ASK) 1. EMBO J. 17, 2596–2606
31 The Arabidopsis Genome Initiative (2000)
Analysis of the genome sequence of the flowering
plant Arabidopsis thaliana. Nature 408, 796–815
32 Kobe, B. and Deisenhofer, J. (1995) Proteins with
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33 Li, J. and Chory, J. (1995) A putative leucine-rich
repeat receptor kinase involved in brassinosteroid
signal transduction. Cell 90, 929–938
34 He, Z. et al. (2000) Perception of brassinosteroids
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35 Takayama, S. et al. (2001) Direct ligand-receptor
complex interaction controls Brassica self-
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Note added in proof
During the final editing of this manuscript, Takayama
et al. reported that Brassica self-incompatibility is
regulated by the direct interaction between SCR
(SP11) and SRK–SLG receptor complex35.

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
a decade ago, considerable advances have been made in the study of plant
membrane transport, but we still do not understand transport regulation.
The genes encoding the transport systems in the various cell membranes are
still to be identified, as are the physiological roles of most transport systems.
A wide variety of complementary strategies are now available to study
transport systems in plants, including forward and reverse genetics,
proteomics, and in silico exploitation of the huge amount of information
contained in the completely known genomic sequence of Arabidopsis.
Plant membrane transporters (pumps, carriers and
channels) participate in various functions including
mineral nutrition, carbon and nitrogen metabolism,
cell signaling, osmoregulation, cell homeostasis,
storage and stress responses. Until recently, although
there was a huge amount of functional data analysing
transporter activities in plant cells and their fine
tuning by a variety of regulation mechanisms,
information about the molecular bases of the
transport processes was scarce. The past decade has
seen tremendous advances in the field of plant
membrane transport. On the one hand, the advent of
molecular techniques and the use of heterologous
expression systems have allowed the identification of
transporter genes and the characterization of the
encoded proteins, providing the first pictures of
the molecular structures of plant transporters1,2. On
the other hand, forward and reverse genetic approaches
have enabled the physiological roles of transport
systems in planta to be unraveled in a few cases.
A new era has begun with the sequencing of
Arabidopsis3. About 18% of the 25 498 predicted
proteins appear to contain two or more membrane
spans, whereas 5–10% represent transport proteins4.
Putative transport functions have been assigned to
these proteins based on homology with already
identified counterparts in other organisms. However,
plant cells might have evolved unique transport
properties, and a major challenge remains to uncover
plant-specific transport systems. Another challenge
arises from the highly compartmentalized
organization of plant cells and the expected
specialized transport equipment of each membrane
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
characterize genes encoding plant transport systems,
and to try to make sense out of the emerging
complexity of transport regulatory networks.
Plant transporters in heterologous systems: how far
can we go?
A major breakthrough in our knowledge of
membrane transport in plants occurred with the use
of heterologous expression systems for cloning
membrane transport genes and/or characterizing the
functional properties of the corresponding proteins.
Several cloning strategies have been described to
isolate membrane proteins based on the detection of
proteins expressed at the surface of host cells.
Immunoselection of membrane proteins has been
performed in both animal cells (COS cells from
green monkey kidney)5,6 and E. coli7. Other systems
have been developed, both in COS cells and in
yeast, based on the detection of targeting of a
peptide of known structure or function to the
membrane: the signal sequence trap8,9. However,
these topology-based screening strategies seem to be
more efficient for secreted and type-I membrane
proteins, with the multiple-transmembrane-segment
proteins – transporters belong mainly to this last
type – being rarely selected.
Although E. coli is the easiest expression system
for protein production, it has been little used in the
membrane protein field because incorrect folding and
targeting of hydrophobic eukaryotic proteins is
common. Many overexpressed membrane proteins
seem to be toxic or expressed as unfolded inactive
proteins. Only a few reports describe the functional
expression of a membrane protein10–12 and E. coli has
mainly been used to determine the membrane
topology of proteins.
Among the heterologous systems used, the
unicellular yeast Saccharomyces cerevisiae has
Hélène Barbier-Brygoo proved to be the most powerful tool, mainly because
Institut des Sciences du many strains defective in specific transport activities
Végétal, UPR 2355, CNRS, are available. Most of our current knowledge about
91198 Gif-sur-Yvette
Cedex, France.
the molecular basis of plant transport has been
e-mail: gained using functional complementation strategies
brygoo@isv.cnrs-gif.fr on yeast mutants or strains with modified metabolic
Frédéric Gaymard pathways. In the early 1990s, an invertase-deficient
Laboratoire de Biochimie mutant unable to hydrolyse extracellular sucrose was
et Physiologie engineered to express an intracellular sucrose
Moléculaire des Plantes,
UMR 5004 (CNRS/INRA/
synthase gene, and several sucrose transporter
ENSA-M/Université cDNAs were cloned using this strain. Later, yeast
Montpellier 2), INRA, strains with defective K+-uptake systems were
34060 Montpellier Cedex 1,
complemented by Arabidopsis cDNA libraries,
France.
which enabled two K+ channel cDNAs to be isolated.
Norbert Rolland After these first successes, yeast mutants in various
Jacques Joyard
Laboratoire de
transport systems were used to clone the
Physiologie Cellulaire corresponding plant genes using this effective
Végétale, UMR 5019 strategy13–17. However, expression of a foreign protein
(CNRS/CEA/Université
in yeast can interfere with its cellular physiology and
Joseph Fourier),
CEA-Grenoble, 38054 the functional complementation might not be due to a
Grenoble Cedex 9, France. replacement of the deficient transport capacity of the
http://plants.trends.com
mutant by the expression of an heterologous
transporter18–20. Thus, functional complementation
of a strain defective in a specific transport pathway
does not necessarily demonstrate that the cloned
gene encodes a plant transport system.
Xenopus oocytes and other animal cells such as
insect or COS cells have mainly been used for
studying the functional properties of cloned channels
and transporters16. Xenopus oocytes were also used
in cloning strategies enabling the identification of
several animal channels and transporters21.
Surprisingly, in spite of a report describing the
expression of a maize K+ channel after the injection
of cRNA in oocytes22, this strategy has not been used
successfully to isolate plant cDNAs.
Yeast allowed the expression cloning of many plant
transport systems, mostly residing in the plasma
membrane of their native cells, whereas both plasma
membrane and tonoplast proteins could be
functionally expressed in Xenopus oocytes. A great
limitation of both these systems is that few (if any)
membrane proteins from organelles (chloroplast and
mitochondria) came out of these functional screens.
The main reason might be the presence of a transit
peptide preventing the protein from reaching the
plasma membrane of yeast cells or oocytes, where its
activity could be detected. In some instances, once a
gene has been identified, no transport activity
corresponding to the expressed protein can be
detected after expression in heterologous systems. In
other cases, such as stomatal guard cells, pronounced
differences have been observed between the activity
of the native K+ channels and that of cloned
α-subunits expressed in Xenopus oocytes16. Several
hypotheses can account for these negative results.
The plant protein might be incorrectly matured or be
targeted to a membrane compartment that is
different from the plasma membrane. Alternatively,
the transport system might require plant-specific
protein partners that are not available in the
heterologous system; for example, if the functional
transport unit results from the heteromeric assembly
of different subunits23. In addition, cell-type-specific
post-translational modifications and/or the presence
of cytosolic compounds might contribute to transport
activity or regulation. In this context, even when a
transport activity can be detected upon heterologous
expression, caution must be exercised in directly
transposing the data to speculate about the
transporter functions in its native cell.
Classical (forward) genetics: which screens?
The isolation of plant mutants altered in specific
transport pathways is potentially a powerful way to
identify genes involved in ion or metabolite transport.
Screens might include the search for mutants altered
in their growth response to exogenous ions24, over- or
underaccumulating specific ions25,26, or mutants
altered in their sensitivity to compounds inhibiting
transport protein activities27,28. Apart from isolating
Review TRENDS in Plant Science Vol.6 No.12 December 2001
many mutants and physiologically characterizing
them, genetic approaches for identifying transport
protein genes have rarely been successful. Only two
cases have been reported to date, which led to the
identification of a nitrate uptake system29 and of a
protein homologous to the Na+–H+ antiporters found
in fungi and animals30. Surprisingly, there are more
examples in the literature of mutants being selected
on the basis of altered developmental processes that,
when the mutated genes were cloned, turned out to
involve transport systems31–34. For instance, the dnd1
(defense, no death 1) mutant fails to produce a
hypersensitive response and exhibits constitutive
systemic resistance and elevated levels of salicylic
acid. DND1 was shown to be a cyclic-nucleotide-gated
channel of previously unknown in planta function32.
These examples show that, in some cases, genetic
strategies based on functional screens related to
transport activities can lead to the identification of
key steps in transport pathways. However, such a
strategy requires a huge amount of work and has led
to the identification of few transporters. In addition,
although it is based on transport processes, such
screening can lead to the isolation of mutations with
pleiotropic effects or to the identification of genes
involved in the regulation of ion homeostasis rather
than in transport steps. In other cases, transporter
genes have been isolated by accident because there
was no way to predict that the selected developmental
phenotype would be associated with a mutation in a
transporter gene. These two types of statements do
not argue in favor of the use of forward genetics as the
most appropriate approach to target transporter
genes specifically.
Identifying plant transporters using proteomics
The use of proteomics as a method to identify all
cellular proteins is currently in a rapid state of
development, and its use to identify transport
proteins is just emerging2,35,36. Two main features of
membrane transporters make them difficult to
identify using proteomics. First, transporters are
often minor components in cell membranes. Their
low abundance can impede their identification and
analysis in plant tissues even by sensitive techniques
such as mass spectrometry2,35. A solution to this
problem is to analyze subcellular proteomes starting
from isolated membranes and vesicles. Second, most
transporters contain a series of 15–25 nonpolar
amino acids constituting transmembrane α-helices
buried in the hydrocarbon core of the membrane.
Such proteins are therefore highly hydrophobic and
are under-represented in two-dimensional (2D)
patterns obtained by polyacrylamide gel
electrophoresis of membranes37. This under-
representation is due (at least in part) to the
currently available proteomic technology, which
favors the specific loss of the most hydrophobic
membrane proteins. This is true for all membranes
unless intrinsic proteins were extracted from the
http://plants.trends.com
579
membrane and solubilized with either organic
solvents35 or nonionic or zwitterionic detergents38
before electrophoresis. However, of the Arabidopsis
plasma membrane proteins that have been
separated by 2D PAGE after solubilization with
various detergents (e.g. C8Ø, ASB14), only a few are
transport proteins: an aquaporin (PIP, monomer and
dimer) and the H+-ATPase, which have six and ten
transmembrane domains, respectively, and (from
roots) a high-affinity nitrate transporter (NRT2)
with 12 transmembrane domains38.
Using chloroplast envelope membranes as a
model, the solubility of hydrophobic proteins in
organic solutions has been used to extract them
from purified membrane fractions35 (Fig. 1).
Polypeptides were separated by one-dimensional
SDS-PAGE, analyzed by tandem mass
spectrometry microsequencing and characterized
by comparison with protein sequences deduced
from genomic sequences present in the databases
(see below). Most of the envelope polypeptides
soluble in chloroform–methanol mixture had
1–13 putative transmembrane α-helices35. The
propensity of hydrophobic proteins to partition into
a chloroform–methanol mixture was directly
correlated to the Res:TM ratio (the ratio of the
number of amino acid residues to the number of
putative transmembrane regions), with a cut-off
value of ~100. For instance, two important
envelope membrane transporters were identified
in the chloroform–methanol-soluble fraction:
the malate–oxoglutarate translocator
(12–13 transmembrane α-helices, Res:TM ratio of 38)
(Fig. 1) and the phosphate–triose-phosphate
translocator (6–7 transmembrane α-helices,
Res:TM ratio of 49). In addition, this fraction
contains a series of putative transporters with
molecular masses ranging from 10 kDa to 100 kDa
and contains one or more putative membrane-
spanning domains. Such proteins do not
correspond to any previously identified chloroplast
envelope transporters but have homologies to
several prokaryotic transporters35. Thus,
chloroform–methanol selective extraction is a
versatile method to recover minor, highly
hydrophobic proteins from purified membrane
systems35,39. However, the proteins that might
correspond to transport systems must still be
identified from among these hydrophobic proteins.
This question should be first addressed in silico.
Bioinformatics: linking genomics and functional analysis
Large-scale genome sequencing efforts, such as the
Arabidopsis Genome Project3 (Box 1), and the resulting
massive amount of data provide the basis for computer-
aided analyses to predict all the open-reading-frames
to be encoded by these genomes. General information
is mostly concentrated on web sites dedicated to
genome annotations and displaying information on
predicted proteins from several plant species. One way
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
is the purification of membranes from plant tissues or cells. The
envelope membranes must be properly characterized and the level of
their purity thoroughly determined35. In Step 2, the hydrophobic proteins
are extracted from envelope membranes with a mixture of chloroform and
methanol35. Envelope polypeptides are then separated by one-dimensional
SDS-PAGE (a), instead of two-dimensional PAGE, to eliminate the risk of loss
of hydrophobic proteins during electrophoresis. In addition, SDS is efficient
at solubilizing highly hydrophobic proteins. Furthermore, most chloroplast
envelope transporters identified to date are highly basic proteins and are
only poorly resolved in the best cases, when using commercially available
immobilized pH gradients. A small proportion (5–10%) of the total envelope
polypeptides is soluble (S) in chloroform–methanol, whereas most
membrane polypeptides remain in the chloroform–methanol-insoluble
phase (I). Polypeptides separated by SDS-PAGE are digested by trypsin (in-
gel digestion) and the peptides then analyzed by tandem mass spectrometry
(MS/MS) for microsequencing (b) or by matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF) to obtain peptide
mass fingerprints (c). The poor resolution of one-dimensional PAGE is
compensated for by the high sensitivity and remarkable ability of MS/MS
to characterize small quantities of proteins, even in complex mixtures.
(b) Proteins can be identified by comparison of peptide sequence tags
with protein sequences deduced from genomic sequences present in the
databases. The protein sequence shown is that of the malate–oxoglutarate
translocator, a major chloroplast envelope transporter of 45 kDa (Ref. 40).
Amino acids in blue correspond to the chloroplast targeting sequence; those
in red correspond to the sequences obtained by MS/MS. Underlined amino
acids correspond to the 12–13 putative transmembrane α-helices. (c) The
circles above the peptide mass fingerprint represent masses of some tryptic
fragments of the envelope malate–oxoglutarate translocator, whereas
the X represents the masses of some trypsin peptides. (d) Hydropathy
profile of the chloroplast envelope malate–oxoglutarate translocator.

http://plants.trends.com
 Review TRENDS in Plant Science Vol.6 No.12 December 2001
Box 1. The global bioinformatic analysis – from the Arabidopsis thaliana
genome sequence to the prediction of plant membrane transporter families
Arabidopsis thaliana genome sequencing: The Arabidopsis Genome
Initiative (AGI) http://www.arabidopsis.org/
Cold Spring Harbor Sequencing Consortium (CSHSC)
http://nucleus.cshl.org/protarab/
European Scientists Sequencing Arabidopsis (ESSA)
http://mips.gsf.de/proj/thal/proj/proj_overview.html
Genoscope-EU Consortium
http://www.genoscope.cns.fr/externe/English/Projets/Projet_S/S.html
Kazusa DNA Research Institute http://www.kazusa.or.jp/kaos/
SPP Consortium http://sequence-www.stanford.edu/ara/SPP.html
The Institute for Genomic Research (TIGR) http://www.tigr.org/
Plant gene predictions
NetPlantGene (splice site prediction)
http://www.cbs.dtu.dk/services/NetPGene/
GeneMark (coding probability of single exons)
http://genemark.biology.gatech.edu/GeneMark/gm_info.html
XGrail (coding probability of single exons) http://compbio.ornl.gov/Grail-1.3/
GENSCAN (gene modeling)
http://genome.dkfz-heidelberg.de/cgi-bin/GENSCAN/genscan.cgi
GENEFINDER (gene modeling)
http://mips.gsf.de/proj/thal/proj/genefinder.html
TRNAscan-SE (detection of transfer RNA genes)
http://www.genetics.wustl.edu/eddy/tRNAscan-SE/
Plant genome annotation (general)
MATDB (MIPS Arabidopsis thaliana database) data of the AGI compiled,
analyzed, annotated and stored http://mips.gsf.de/proj/thal/db/index.html
Center for Medicago Genomics Research
http://www.noble.org/medicago/index.htm
ZmDB (Zea mays DataBase) http://www.zmdb.iastate.edu/
CSHL (maize genome analysis) http://nucleus.cshl.org/maizegenome/
CSHL (rice genome analysis) http://nucleus.cshl.org/riceweb/
TIGR (The Institute for Genomic Research) (includes gene index pages for
12 plant species) http://www.tigr.org/tdb/tgi.shtml
TOGA (TIGR Orthologous Gene Alignment database) (Includes orthologs
from Arabidopsis, rice, tomato, potato, Medicago, soybean, maize,
wheat) http://www.tigr.org/tdb/toga/toga.shtml
Functional libraries including plant transport systems
MATDB tables (functional categories)
http://mips.gsf.de/proj/thal/db/tables/tables_func_frame.html
AMPL (Arabidopsis Membrane Protein Library)
http://www.cbs.umn.edu/arabidopsis/
Genomic comparisons of membrane transport systems
http://www-biology.ucsd.edu/~ipaulsen/transport/
PlantsT (functional genomics of plant transporters) http://plantst.sdsc.edu/
P-type ATPase database http://biobase.dk/%7Eaxe/Patbase.html
Gene families encoding aquaporins
http://mbclserver.rutgers.edu/CPGN/AquaporinWeb/Aquaporin.group.html
of making sense of the huge amount of data that is
being generated by high-throughput DNA technology
is to get information on the predicted proteins.
Several web sites displaying information on
predicted plant transporter families have recently
http://plants.trends.com
581
been created (Box 1). The ‘manually assigned
functional catalog’ of the MATDB site provides
tables and catalogs of functional categories,
including transporter families, for the hypothetical
Arabidopsis proteins. The AMPL is a collection of
almost 4600 predicted polytopic Arabidopsis
membrane protein sequences (containing at least
two predicted membrane-spanning domains) that
have been clustered into families based on sequence
homology4, a subsection being dedicated to
Arabidopsis transporter families (‘Arabidopsis
transporter view’). The ‘Genomic comparisons of
membrane transport systems’ site contains the
distribution of known and putative polytopic
membrane transport proteins for all organisms for
which completely sequenced genomes are available.
Transport systems for each organism were
classified according to putative membrane topology,
protein family, bioenergetics and substrate
specificity. Complete lists of the transporters from
each organism are provided. The ‘Transport
proteins in Arabidopsis’ page displays graphical
data about almost 900 classified Arabidopsis
transporters. Recently, huge efforts have been
made to analyze specific subfamilies of plant
transporters. Inventories and phylogenic
relationships within subfamilies of Arabidopsis
cation transporters41, ABC proteins42, P-type
pumps43, MIPs (Ref. 44) and maize aquaporins45
have recently been published, and corresponding
Web sites have been created (Box 1).
The first step after identifying a new protein
sequence is the search for homology between the
sequence and those from various databases (Box 2;
Fig. 2). This is often the simplest way to obtain
functional information when it is available. However,
functional annotation of a sequence through
comparisons encounters a major limitation: almost
half of the proteins predicted in Arabidopsis are
unrelated to any protein with a known function46, and
functional information is still lacking for 60% of the
Arabidopsis membrane protein sequences4.
When the sequence of an unknown protein is too
distantly related to any protein of known structure
to detect its resemblance by overall sequence
alignment, relationships can be revealed by the
occurrence of a particular cluster of residue types in
its sequence (Box 2). The use of protein sequence
patterns, profiles, motifs, signatures or fingerprints
to determine the function of proteins offers an
alternative to more classical alignment programs47.
These databases help to determine which known
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
based on principles of probabilistic modeling.
Examples of such methods include the use of
probabilistically derived score matrices to determine
the significance of sequence alignments, the use of
hidden Markov models as the basis for profile
582 Review TRENDS in Plant Science Vol.6 No.12 December 2001
Box 2. The reverse bioinformatic analysis – from a unique sequence to
the prediction of a plant membrane transporter
Homology search tools
BLAST (NCBI Web Site) queries performed on non redundant or species-
specific databases (includes Arabidopsis, rice, maize)
http://www.ncbi.nlm.nih.gov/BLAST/
TAIR (The Arabidopsis Information Resource) http://www.arabidopsis.org/
Protein families predictions
PFAM (database of protein domain families) http://www.sanger.ac.uk/Pfam/
PRODOM (protein domain database)
http://protein.toulouse.inra.fr/prodom.html
BLOCKS (blocks database) http://www.blocks.fhcrc.org/about_blocks.html
PRINTS (protein motif fingerprint database)
http://www.bioinf.man.ac.uk/dbbrowser/PRINTS/
PROSITE (dictionary of protein sites and patterns)
http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html
Hidden Markov models for profile predictions
MEME (multiple EM for motif elicitation for discovering motifs in a group
of related protein sequences) http://meme.sdsc.edu/
HMMER (profile hidden Markov Models for biological sequence analysis:
provides statistical models of the primary structure consensus of a
protein family) http://hmmer.wustl.edu/
Prediction of helical transmembrane proteins
TMpred (prediction of membrane-spanning regions and orientation based
on the statistical analysis of a database of naturally occurring
transmembrane proteins)
http://www.ch.embnet.org/software/TMPRED_form.html
PSORT Prediction (recognition of most probable transmembrane segments
from the average hydrophobicity value of 17-residue segments)
http://psort.nibb.ac.jp/helpwww.html#src
Physico-chemical profiles (protein sequence analysis for displaying the
hydropathic character of a protein) http://pbil.univ-lyon1.fr/
TMAP (prediction from multiple sequence alignment)
http://www.mbb.ki.se/tmap/
SOSUI (classification and secondary structure prediction of membrane
proteins) http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html
HMMTOP (hidden Markov model for topology prediction of helical
transmembrane proteins) http://www.enzim.hu/hmmtop/index.html
PHD (suite of programs predicting 1D structure from multiple sequence
alignments. PHDhtm predicts the location and topology of
transmembrane helices) http://dodo.cpmc.columbia.edu/predictprotein/
Subcellular localization (compatible with plant input sequences)
ChloroP v1.1 (chloroplast transit peptide prediction)
http://www.cbs.dtu.dk/services/ChloroP/
TargetP v1.01 (prediction of subcellular location)
http://www.cbs.dtu.dk/services/TargetP/
Predotar (prediction of putative mitochondrial and plastid targeting
sequences) http://www.inra.fr/Internet/Produits/Predotar/
PSORT Prediction (Prediction of subcellular location)
http://psort.nibb.ac.jp/helpwww.html#src
searches to identify distant members of sequence
families (Box 2) and the inference of phylogenetic
trees using maximum likelihood approaches48.
http://plants.trends.com
Efficient tools are also available for other
analyses. The function of a protein is tightly
correlated with its subcellular localization, which is
linked to its physicochemical nature. There are tools
to predict transmembrane domains in protein
sequences (Box 2) or protein sorting signals, signal
peptide or cleavage sites (Box 2). Some methods are
based on the hydrophobicity plot of the analyzed
protein (e.g. physicochemical profiles) or on
comparisons to a transmembrane database
(e.g. TMpred), whereas others predict the location
and topology of transmembrane helices from multiple
sequence alignments (e.g. TMAP, PHDhtm). A hidden
Markov model was recently developed to predict the
topology of helical transmembrane proteins
(HMMTOP). This method is based on the hypothesis
that the localization of the transmembrane
segments and their topology are determined by the
difference in the amino acid distributions in various
structural parts of these proteins rather than by
specific amino acid compositions of these parts.
However, this apparently obvious approach is not
always satisfactory, and information obtained by
predicting sorting signals is not reliable. Gene
assignments are often unreliable in their 5′ regions49,
the analyses of which are usually the basis of the
prediction of protein sorting signals. Other
limitations might exist: for instance, porins present
in outer membranes from plastids and
mitochondria50 do not contain any cleavable (and
therefore predictable) N-terminal transit sequence
or any transmembrane α-helices.
Reverse genetics: a necessary gateway to integrated
transport function
In spite of the growing amount of information on
protein sequences and functional characterization,
the fundamental question about the physiological role
in planta remains to be clarified for most transport
systems. The isolation and characterization of loss-of-
function mutants is a powerful way to assign a
biological function to a known gene. In many model
organisms (yeast, bacteria, mice), homologous
recombination can be used efficiently to target
mutations to specific genes. In plants, homologous
recombination has been described but appears to be
difficult to implement and is not feasible on a large
scale, except for the moss Physcomitrella patens51.
An alternative, highly efficient procedure for
isolating mutants in identified genes is insertional
mutagenesis. The improvement of plant
transformation techniques52 has led to the formation
of large collections of plants mutagenized by T-DNA,
insertion elements or transposons53,54. Several
collections of plants or DNA pools and databases of
flanking sequence tags (databases of systematic
sequencing of insertion sites) can be screened for the
identification of lines harboring insertions within a
gene of interest. Moreover, recent reports of RNA-
mediated interference have opened new prospects for
 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

http://plants.trends.com
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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