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Plant, Cell and Environment (2000) 23, 175–184
AtSTP3, a green leaf-specific, low affinity monosaccharideH+ symporter of Arabidopsis thaliana
M. BÜTTNER,1 E. TRUERNIT,1 K. BAIER,2 J. SCHOLZ-STARKE,1 M. SONTHEIM,1 C. LAUTERBACH,1 V. A. R. HUSS1
& N. SAUER1
1
Universität Erlangen-Nürnberg, Lehrstuhl Botanik II, Molekulare Pflanzenphysiologie, Staudtstrasse 5, D-91058 Erlangen,
Germany and 2Humbold-Universität zu Berlin, Institut für Biologie, Biochemie der Pflanzen, Chausseestraße 117, D-10115
Berlin, Germany
ABSTRACT
This paper describes the expression analyses of the AtSTP3
gene of Arabidopsis thaliana, the functional characterization of the encoded protein as a new monosaccharide transporter, and introduces the AtSTP gene family. The kinetic
properties of the AtSTP3 protein (for sugar transport
protein 3) were studied in a hexose transport deficient
mutant of Schizosaccharomyces pombe. AtSTP3 represents a new monosaccharide transporter that is composed
of 514 amino acids and has a calculated molecular mass of
55·9 kDa. Kinetic analyses in yeast showed that AtSTP3 is
a low affinity, energy-dependent H+ symporter with a Km
for d-glucose of 2 mm. RNase protection analyses revealed
that AtSTP3 is expressed in leaves and floral tissue of Arabidopsis. This expression pattern of the AtSTP3 gene was
confirmed in AtSTP3 promoter-b-glucuronidase (GUS)
plants showing AtSTP3-driven GUS activity in green
leaves, such as cotelydons, rosette and stalk leaves and
sepals. Wounding caused an induction of GUS activity in the
transgenic plants and an increase of AtSTP3 mRNA levels
in Arabidopsis wild-type plants. Polymerase chain reaction
analyses with degenerate primers identified additional new
AtSTP genes and revealed that AtSTP3 is the member of a
large family of at least 14 homologous genes coding for
putative monosaccharide-H+ symporters (AtSTPs).
Key-words: Arabidopsis; AtSTP3; gene family; glucose;
monosaccharide transport.
INTRODUCTION
In Arabidopsis newly synthesized sucrose is loaded into the
companion cells of the phloem by the sucrose-H+ symporter
AtSUC2 (Sauer & Stolz 1994; Truernit & Sauer 1995;
Stadler & Sauer 1996) and allocated from the photosynthetically active source tissues towards the carbon importdependent sink tissues, such as roots, flowers or siliques.This
allocation has been analysed with exogenously supplied fluCorrespondence: N. Sauer, Botanik II, Molekulare Pflanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D91058 Erlangen, Germany. Fax: +49 9131 85 28751; e-mail:
[email protected]
© 2000 Blackwell Science Ltd
orescent compounds (Oparka et al. 1994) and most recently
with transgenic plants expressing the gene of the jelly fish
green fluorescent protein (GFP) under the control of the
companion cell-specific AtSUC2 promoter (Imlau, Truemit
& Sauer 1999; Oparka et al. 1999). The concurring result of
these experiments was that both the small carboxyfluorescein (CF) molecule (Oparka et al. 1994) as well as the
27 kDa GFP are allocated with the stream of assimilates and
symplastically unloaded from the phloem into sink tissues,
such as petals, anthers, ovules or the elongation zone of
roots (Imlau et al. 1999). Inside these sink tissues CF and the
GFP can traffic cell to cell, thereby demonstrating the existence of plasmodesmata with large-size exclusion limits
(Imlau et al. 1999). Although not directly shown it is likely
that the allocation of assimilates, such as sucrose or amino
acids, from the source leaf companion cells into the cells of
different sink organs can follow the same symplastic pathways. Apoplastic transport of sucrose is unlikely to play a
major role between the cells of these tissues.
However, numerous cells and tissues are not symplastically connected to the phloem. CF applied to leaves and
symplastically unloaded from the root protophloem cannot
enter the cells at the very tip of a root nor does it move into
mature root hairs, suggesting that these cells have no access
to symplastically unloaded assimilates (Oparka et al. 1994).
Moreover, no symplastic unloading of CF is seen from the
metaphloem in the fully differentiated parts of the roots
(Oparka et al. 1994). Similarly, GFP that has migrated symplastically into the seed coat of a developing Arabidopsis
ovule cannot traffic into cells of the embryo (Imlau et al.
1999) and also several cell layers in anthers, such as the
middle layer or the tapetum, have been shown to lack symplastic connections (Clément & Audran 1995). These and
other results demonstrate the existence of isolated symplastic domains depending on the carrier-mediated import
of assimilates.
In many plant tissues this step is catalyzed by
monosaccharide-H+ symporters. Glucose and fructose
result from the extracellular hydrolysis of sucrose by cell
wall-bound invertases (Ehneß & Roitsch 1997; Sturm &
Chrispeels 1990) and both, sucrose hydrolysis and monosaccharide import can be modulated developmentally
(Weber et al. 1995; Weber et al. 1997) or in response to envi175
176 M. Büttner et al.
ronmental factors, such as wounding or pathogen infection
(Sturm & Chrispeels 1990; Truernit et al. 1996). Since the
identification of AtSTP1 as the first higher plant monosaccharide-H+ symporter (Sauer, Friedländer & Gräml-Wicke
1990b) several homologous genes and cDNAs have been
cloned by polymerase chain reaction (PCR) or heterologous screening from different plant species (Sauer &
Stadler 1993; Weig et al. 1994; Truernit et al. 1996; Weber
et al. 1997; Ylstra et al. 1998; Truernit et al. 1999). Using functional expression in yeast (Sauer et al. 1990b; Sauer &
Stadler 1993; Weig et al. 1994; Truernit et al. 1996; Weber
et al. 1997; Truernit et al. 1999) or Xenopus oocytes (Boorer,
Loo & Wright 1994) and analyses of the recombinant
protein after reconstitution into proteoliposomes (Stolz
et al. 1994) many of these transporters were characterized
as energy-dependent, high affinity H+-symporters with the
Km values for glucose ranging from 10 to 50 mm. Detailed
localization analyses on the cellular level showed that
indeed many of these monosaccharide transporter genes
are expressed in symplastically isolated cells, such as pollen
grains (Truernit et al. 1996; Ylstra et al. 1998; Truernit et al.
1999), growing pollen tubes (Ylstra et al. 1998), guard cells
(Stadler & Sauer, unpublished) or in the outermost cell
layers of symplastically separated tissues, such as the
embryo (Weber et al. 1997). Typically, the expression of one
specific monosaccharide transporter gene is confined to
certain cells and to a limited time during development,
which may explain the large number of putative monosaccharide transporter genes found in different plant species
(Roitsch & Tanner 1994; Weig et al. 1994).
In this article AtSTP3, a new monosaccharide transporter
of Arabidopsis with unusual properties, is described.
AtSTP3 represents a low affinity H+-symporter with a Km
for d-glucose of 2 mm. The AtSTP3 gene is not expressed
in a sink tissue but rather in all green Arabidopsis leaves.
The possible function of this transporter is discussed. Furthermore, experiments are presented describing the AtSTP
gene family of Arabidopsis.
ing the C-terminal 105 amino acids of AtSTP1 was used to
screen 128 000 plaque forming units of a cDNA library
from Arabidopsis thaliana strain Columbia wild type
(obtained from Andreas Bachmaier, Max-Planck-Institut
für Züchtungsforschung, Köln, Germany) at low stringency
(Sauer et al. 1990b). After low stringency washes with
2 ¥ SSC at 37 °C (1 ¥ SSC = 0·15 m NaCl, 0·015 m sodium
citrate) and exposure on Kodak-X-Omat AR film (Kodak,
Rochester, NY, USA) the filters were washed at high stringency (0·1 ¥ SSC, 50 °C) and exposed a second time. For
one lambda plaque (l113) a positive signal was obtained
only after the washes at low stringency. This signal was lost
after the high stringency washes. The EcoRI insert of l113
was subcloned into pUC19 (pKF113), sequenced and identified as a partial cDNA clone of a new putative monosaccharide transporter (AtSTP3). Screenings of an additional
3·1 million plaque forming units resulted in 39 independent
AtSTP3 cDNAs. Only one clone (pKB39) contained the
entire coding sequence of AtSTP3, however, it represented
an unspliced mRNA precurser with one intron. Another
cDNA clone (pKF20) lacked the first adenine residue of the
start codon. Using a unique AlwNI site close to the 5¢-end
of the pKB39 coding sequence an intron-less cDNA
(pSTP3) was constructed from pKB39 and the partial
cDNA clone pKF20. A second complete, intron-less cDNA
was generated from pKF20 in a PCR adding this missing
adenine residue. The resulting sequence had EcoRI ends,
was cloned into pUC19 (pSTP3-PCR) and sequenced
(Sanger, Nicklen & Coulson 1977).
To obtain further homologous AtSTP sequences and to
get a rough estimate of the size of the AtSTP gene family
in Arabidopsis degenerate oligonucleotide primers (Weig
et al. 1994) were used for PCR analyses (ATH1: 5¢-GGW
TTY GCW TGG TCW TGG GGW CC–3¢; ATH3: 5¢-WGG
DAT WCC YTT DGT YTC WGG–3¢). Amplified
fragments were blunted and cloned into SmaI-digested
pUC19. Sequence analyses of 46 subcloned PCR fragments revealed a gene family comprising at least 11 putative monosaccharide transporters in Arabidopsis thaliana
(AtSTP1 to AtSTP11).
MATERIALS AND METHODS
Strains
Isolation of AtSTP3 genomic sequences
Arabidopsis thaliana C24 was grown in the greenhouse or
on agar medium as previously described (Truernit & Sauer
1995). Escherichia coli strain DH5a (Hanahan 1983) was
used for cloning, Schizosaccharomyces pombe strain YGS5 (kindly provided by B. Milbrad and M. Höfer, University
of Bonn, Germany) was used for heterologous expression.
Transformations of Arabidopsis thaliana were performed
with Agrobacterium tumefaciens strain LBA4404 (Ooms
et al. 1982).
Screening of a genomic library of Arabidopsis (strain
Columbia wild type in lGEM11; generated by J.T.
Mulligan and R.W. Dawis, Stanford, CA; supplied by the
EEC Arabidopsis stock centre, Köln, Germany) with radiolabelled sequences of pSTP3 yielded the positive clones
lK1 and lK2. After DNA gel-blot analysis with a radiolabelled 246-bp fragment from the 5¢-end of pKF20 a 6 kb
PstI fragment of lK1 was subcloned into pUC19, yielding
the plasmid pET30.
Isolation of a full length AtSTP3 cDNA and of
partial sequences of AtSTP5 to AtSTP11
Functional characterization of AtSTP3 by
heterologous expression
A radiolabelled 370-bp ApaI/HindIII fragment of the
genomic AtSTP1 clone pUGW7 (Sauer et al. 1990b) encod-
The insert of the PCR-generated AtSTP3 cDNA clone
pSTP3-PCR was ligated into the unique EcoRI site of the
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
A family of monosaccharide transporters in Arabidopsis 177
S. pombe/E. coli shuttle vector SAP-E (Truernit et al. 1996).
Constructs with the insert in sense or antisense orientation
were used for transformation (Ito et al. 1983) of S. pombe
strain YGS-5 that carries a mutation in the endogenous
hexose transport system.
Wounding of Arabidopsis leaves
Rosette leaves from sterile Arabidopsis leaves were
wounded with a scalpel to give 4 to 5 mm sections and
incubated in 5 mm NaPO4 buffer, pH 5·5 as described by
Corbin, Sauer & Lamb (1987). At the indicated time points,
material was withdrawn and frozen in liquid nitrogen.
RNA isolation, RNA gel blot analysis, and
RNase protection assay
Total RNA was isolated as described by Sauer et al. (1990a).
RNase protection assays were performed according to the
protocol of Ratcliffe et al. (1990), with the modifications
described by Gahrtz et al. (1996).
For the AtSTP3 probe a 224 bp StuI/PvuII fragment of
pSTP3 was cloned into EcoRV-digested pBluescript II SK
(Stratagene, San Diego, CA, USA) in such a way that antisense RNA could be generated with T7 RNA polymerase
after linearization with EcoRI (pRPA32). The hybridizing sequence of the resulting antisense probe was 224
nucleotides long.
For the AtSUC2 probe a 217 bp HincII fragment of
pTF2035 (Sauer & Stolz 1994) was cloned into HincIIdigested pSP64 (Promega Corporation, Madison, WI,
USA), excised again with HindIII and BamHI and cloned
into pBluescript II SK-(Stratagene, San Diego, USA) in
such a way that antisense RNA could be generated with
T3 RNA polymerase after linearization with HindIII
(pRPA201). The hybridizing sequence of the resulting antisense probe was 217 nucleotides long.
The probes for AtSTP4 mRNA and for the 18S rRNA
control were the same as described (Truernit et al. 1996).
Localization of AtSTP3 expression by reporter
gene analyses
An 1817 bp AlwNI fragment (EMBL accession No.
AJ001363) that covered primarily upstream promoter
sequences and that encoded the first 15 amino acids of
AtSTP3 was excised from pET30, blunted with mung bean
nuclease and ligated into XbaI-digested and Klenowtreated pUC19. The insert of this construct was excised with
BamHI/SalI and ligated into the respective sites of pBI101
yielding pET101STP3. Transformation of Arabidopsis with
pET101STP3 resulted in 35 independent transformants.
Reporter gene activity was studied in 10 of these plants in
the T1 generation.
Transformation of Arabidopsis and tobacco
Arabidopsis root explants were transformed with Agrobacterium harbouring the construct pET101STP3 according
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
to the protocol of Valvekens, Van Montagu & Van
Lijsebettens (1988) with the modifications described
(Truernit & Sauer 1995). Tobacco leaf discs were transformed with the same strain of Agrobacterium as described
(Horsch et al. 1985).
Sequence data analysis
Protein sequences were submitted to the BCM Search
Launcher (Human Genome Center, Baylor College of
Medicine, Houston TX, HTTP://kiwi.imgen.bcm.tmc.edu:
8088/search-launcher launcher/html) for automated
multiple alignement using the Clustal W 1·7 program
(Thompson, Higgins & Gibson 1994) under standard parameters. Regions of length variability (mainly at the N- and
C-termini) were excluded from further analyses resulting in
472 amino acid residues that were used for the analyses of
AtSTP sequences. Neighbour-joining (NJ) and maximum
parsimony (MP) bootstrap analyses were conducted with
the phylip package 3·572c of Felsenstein (1995). The
Kimura formula was selected for calculating the evolutionary distances that served as input for the NJ program. Addition of taxa was jumbled and repeated three times for each
individual bootstrap replication in the MP analysis. Output
tree-files were combined with the consense program and
converted into a graphical representation by the Treeview
program for Macintosh (Page 1996).
RESULTS
Isolation of AtSTP3 sequences
The partial cDNA clone of a new putative monosaccharide
transporter was identified during a low stringency screening of an Arabidopsis cDNA library with a radiolabelled
genomic fragment of the AtSTP1 monosaccharide-H+ symporter gene (Sauer et al. 1990b). The EcoRI insert of the
purified lambda clone (l113) was subcloned into pUC19
yielding pKF113. Sequence analyses revealed significant
homology to previously described cDNAs of Arabidopsis
monosaccharide transporters (Sauer et al. 1990b; Truernit
et al. 1996; Truernit et al. 1999) and the gene was named
AtSTP3 (for Arabidopsis thaliana sugar transport protein 3). Additional screenings of this cDNA library with
the insert of l113 yielded one clone harbouring the complete sequence interrupted by an intron (pKB39) and one
clone lacking the first base of the start ATG (pKF20). This
incomplete ATG in pKF20 was identical to the first ATG
in pKB39 that was in the same reading frame with two
upstream stop codons. Complete, intron-less AtSTP3
cDNAs were generated by ligating appropriate fragments
of pKB39 and pKF20 (yielding pSTP3) or by PCR (yielding pSTP3-PCR) and cloned into the EcoRI site of pUC19.
The AtSTP3 cDNA and the translated protein sequence
are presented in Fig. 1. The open reading frame is 1542 bp
long and encodes a protein of 514 amino acids with a
calculated molecular mass of 55·87 kDa and an isoelectric
178 M. Büttner et al.
point of 10·1. With the AtSTP1, AtSTP2 and AtSTP4 monosaccharide transporters of Arabidopsis this protein shares
56·3, 48·3 and 54·4% identical amino acids, respectively. As
for these other proteins, hydrophilicity analyses predicted
12 transmembrane helices, a typical feature of transporters
belonging to the major facilitator superfamily described by
Marger & Saier (1993). The AtSTP3 protein has one consensus sequence for N-glycosylation (Asn157; Fig. 1) that is
conserved in AtSTP2 (Asn150) and AtSTP4 (Asn150). In
all of these proteins this residue is part of the putative transmembrane helix IV and thus unlikely to be glycosylated.
A data bank search with AtSTP3 cDNA sequences identified a genomic fragment of chromosome V (AB012239)
containing the AtSTP3 gene. Comparison of genomic and
cDNA sequences showed that the AtSTP3 gene is interrupted by two introns of 198 bp and 75 bp. The sequence of
the first intron (198 bp) is identical to the intron sequence
in the above-mentioned cDNA clone of an unspliced
AtSTP3 mRNA precursor. The positions of the introns
(Fig. 1) are conserved among the so-far characterized
Arabidopsis sugar transporter genes.
AtSTP3 encodes a new monosaccharide
transporter
The PCR-generated AtSTP3 cDNA was cloned into the
EcoRI site of the S. pombe/E. coli shuttle vector SAP-E
(Truernit et al. 1996) and expressed in the S. pombe mutant
YGS-5 (Milbradt & Höfer 1994) for functional characterization. AtSTP3 accepts several monosaccharides as substrates (glucose > xylose > 3-O-methylglucose (3-OMG) >
mannose > galactose > fructose) with the relative transport
rates for galactose and fructose being very low (data not
shown). The Km value of AtSTP3 for d-glucose is 2 mm
(mean value of two experiments), which is about 50 to 100fold higher than the Km values determined for the other
Arabidopsis monosaccharide transporters (AtSTP1, 20 mm;
Sauer et al. 1990b; AtSTP2, 50 mm; Truernit et al. 1999;
AtSTP4, 15 mm; Truernit et al. 1996) or the Chlorella
kessleri glucose transporter CkHUP1 (15 mm; Sauer et al.
1990b).
This high Km value might indicate that AtSTP3, unlike
the other plant monosaccharide transporters, is a monosaccharide facilitator rather than an energy-dependent
monosaccharide-H+ symporter. It has been shown previously that monosaccharide transport in the green alga
Chlorella kessleri can switch between an energy-dependent
high affinity state and an energy-independent low affinity
state (Komor & Tanner 1974). The Km value of this transport system for 6-deoxyglucose increased more than 100fold during the switch from the energy-dependent to the
energy-independent state. Moreover, the Km values for the
mammalian glucose facilitators are also in the mM range
(Walmsley et al. 1998). The energy dependence of monosaccharide transport by AtSTP3 was analysed using the
non-metabolizable glucose analog 3-OMG. All results presented in Fig. 2 show that AtSTP3 catalyzes the uptake of
3-OMG in an energy-dependent manner: (i) 3-OMG was
Figure 1. cDNA and derived protein sequences of the AtSTP3
cDNA clone pSTP3. Conserved primer binding sites used for
PCR amplification of putative AtSTP sequences are indicated by
horizontal arrows. The alternative start of the polyA-tail in the
unspliced cDNA clone pKB39 and the positions of the two
introns in AtSTP3 genomic sequences are given. The EMBL
accession number for the AtSTP3 cDNA sequence is AJ002399.
accumulated inside S. pombe cells to a concentration higher
than that in the medium; (ii) uptake rates for 3-OMG were
increased in the presence of the metabolizable substrate
ethanol, and (iii) the presence of carbonyl cyanide-mchlorophenylhydrazone (CCCP), an uncoupler of trans© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
A family of monosaccharide transporters in Arabidopsis 179
Figure 3. RNase protection analysis of AtSTP3 expression in
different tissues. For the analyses with a radiolabelled AtSTP3
probe 20 mg of total RNA from the indicated tissues were used
per lane. For control analyses with an 18S-rRNA probe 5 ng of
total RNA were used per lane.
Expression of AtSTP3 in response to wounding
Figure 2. Functional characterization of recombinant AtSTP3
protein in S. pombe.Transport of the non-metabolizable glucose
analog 3-OMG was determined in S. pombe cells expressing
AtSTP3 under the control of the S. pombe adh promoter without
further additions (), in the presence of 100 mm ethanol () and
in the presence of 100 mm ethanol and 50 mm carbonyl cyanidem-chlorophenylhydrazone (). The initial outside concentration
of 3-OMG in all uptake tests was 0·1 mm. Values above the
broken line exceeded the concentration equilibrium between
intracellular and extracellular 3-OMG.
membrane proton gradients, completely abolished 3-OMG
uptake by AtSTP3. These results suggest that AtSTP3
is a low affinity, energy-dependent monosaccharide transporter, most likely a H+-symporter.
Localization of AtSTP3 gene expression
RNase protection analyses with RNAs from various Arabidopsis tissues were performed to analyse the preferred
sites of AtSTP3 expression (Fig. 3). High AtSTP3 mRNA
levels were detected only in leaves. Small amounts of
mRNA were also identified in floral tissue and even less in
stalks. No mRNA was seen in roots and fruits.
To confirm these results Arabidopsis and tobacco plants
were transformed using Agrobacteria harbouring the
plasmid pET101STP3 (Fig. 4). In the resulting AtSTP3 promoter-GUS plants expression of the b-glucuronidase
(GUS) is driven by the AtSTP3 promoter. Of 35 independent Arabidopsis transformants 10 were analyzed in the T1
generation for GUS activity. In all AtSTP3 promoter-GUS
plants GUS staining could be detected in the green leaves
of all developmental stages, such as cotelydons, rosette
leaves, stalk leaves and sepals (Fig. 5). No GUS staining was
detected in petals, pods or roots of the AtSTP3 promoterGUS plants. These results confirm the RPA data showing
accumulation of AtSTP3 mRNA in leaves of AtSTP3
promoter-GUS plants. The low mRNA levels seen in
mRNA preparation from flowers are most likely due to
AtSTP3 expression in sepals.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
The Arabidopsis AtSTP4 monosaccharide gene has been
shown to be strongly expressed in root tips and pollen
grains and to a much lesser extent also in leaves (Truernit
et al. 1996). The AtSTP4 expression in leaves resembled the
expression pattern described for AtSTP3 in this paper.
AtSTP4 expression was not homogenous. It was totally
absent from some leaves and in others it was shown to
be spotty or cloudy (Truernit et al. 1996). Moreover, expression of AtSTP4 was shown to be strongly induced in
response to wounding, pathogen infection and elicitor challenge (Truernit et al. 1996). We were interested to see, if the
expression of AtSTP3 is also modulated in response to
environmental stress. Analyses of transgenic tobacco plants
transformed with pET101STP3 (Fig. 4) showed that indeed
wounding caused a drastically enhanced GUS histochemical staining in cells directly adjacent to the wounding site
(Fig. 6a).
To confirm this indirect evidence for a wound induction
Figure 4. Restriction map and N-terminal amino acid sequence
of the AtSTP3 promoter-b-glucuronidase fusion construct used
for the transformation of Arabidopsis and tobacco plants.The
start ATG of AtSTP3 and the start ATG of the GUS gene are
shown. The first 25 amino acids of the resulting fusion protein
are presented. Amino acids 1–14 (bold) are from the AtSTP3
protein and are followed by 10 amino acids (italic) that are
derived from polylinker sequences in pBI101. The last amino
acid (marked with an asterisk) represents the start methionine of
b-glucuronidase. The EMBL accession number for the AtSTP3
promoter sequence is AJ001363.
180 M. Büttner et al.
PCR reactions were performed with genomic DNA using
the degenerate oligonucleotide primers ATH1 and ATH2
that had been designed to bind to highly conserved
sequence domains in monosaccharide transporter genes
(Fig. 1; Weig et al. 1994). The inserts of 46 independently
cloned 250-bp PCR fragments were sequenced and compared to the already existing AtSTP sequences.
The results are summarized in Table 1. Besides the
expected fragments of the already known Arabidopsis
monosaccharide transporters AtSTP1, AtSTP2 and
AtSTP4, sequences from seven additional, so far uncharacterized monosaccharide transporters were identified. No
fragment of the AtSTP3 gene described in this paper was
found. This suggests that the result of our PCR search for
homologous AtSTP genes may not yet describe the entire
family, and that other homologous genes may be missing as
Figure 5. GUS histochemical staining in Arabidopsis plants
expressing GUS under the control of the AtSTP3 promoter.(a)
rosette leaf with cloudy GUS staining; (b) flower with GUS
staining in the sepals; (c) stalk section showing GUS staining
only in the green leaves, including the sepals of the developing
flowers; (d) young rosette. All tissues had been cleared with 70%
ethanol. Scale bars are 2 mm.
of AtSTP3 we analysed the effect of wounding on AtSTP3
mRNA levels in Arabidopsis wild-type plants. Figure 6b
shows that expression of AtSTP3 is indeed induced in
response to wounding although it is much slower than the
expression of the previously described AtSTP4 gene
(Fig. 6b). Whereas the maximum of AtSTP4 induction is
reached already after 2 h, strong induction of AtSTP3 is
seen only after 8 h. This effect is specific and the simultaneously analysed mRNA levels of the companion cellspecific AtSUC2 sucrose transporter are not effected by
this treatment. 18S rRNA was used as loading control
(Fig. 6b). Comparison of the first lane in Fig. 6b (mRNA
isolated from rosette leaves of sterile plants) with the first
lane in Fig. 3 (mRNA isolated from rosette leaves of plants
grown on potting soil) reveals similar differences in the
basal expression levels as described for AtSTP4 (Truernit
et al. 1996) and as reflected by the GUS histochemical staining of the rosette presented in Fig. 5d.
How large is the monosaccharide transporter
family in Arabidopsis?
Including AtSTP3, four monosaccharide transporter genes
have been isolated and characterized in Arabidopsis to
date. We were interested to see, if these four genes represent the entire family of Arabidopsis monosaccharide
transporters or if there are further homologous genes
encoding putative monosaccharide transporters. Therefore,
Figure 6. Induction of AtSTP3 promoter activity in response to
wounding. (a) weak, cloudy GUS staining and strong, woundinduced GUS staining (arrow) in the leaf of a transgenic tobacco
plant expressing the GUS reporter gene under the control of the
AtSTP3 promoter. Only the GUS staining along the lesion was
induced by cutting with a razor blade. Scale bar = 1 cm.(b)
RNase protection analyses of the expression levels of various
sugar transporter genes in response to wounding. For the
analyses with radiolabelled sugar transporter probes (AtSTP3,
AtSTP4 and AtSUC2) 15 mg of total RNA were used per lane.
For control analysis with the 18S rRNA probe 4·5 ng of total
RNA were used per lane.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
A family of monosaccharide transporters in Arabidopsis 181
Table 1. Comparison of 14 characterized or putative monosaccharide transporter genes from Arabidopsis
Gene
name
Accession
No.
Amino
acids
Percentage identity
to AtSTP1
Located on
chromosome
Introns at
position
ESTs
found
Functionally
characterized
AtSTP1
AtSTP2
AtSTP3
AtSTP4
AtSTP5
AtSTP6
AtSTP7
AtSTP8
AtSTP9
AtSTP10
AtSTP11
AtSTP12
AtSTP13
AtSTP14
AC007259
AJ001362
AJ012399
AB025631
AJ001658
AJ001659
AJ001660
AF077407
AJ001662
AB025631
AJ001664
AL022603
AF077407
AC004260
522
499
514
514
–
504
513
507
517
514
520
508
542
504
100·0
50·2
56·3
63·2
–
54·0
56·5
53·3
61·7
61·5
61·0
80·5
59·5
53·0
I
–
V
III
–
III
IV
V
I
III
V
IV
V
I
12--5
12--5
12--n.d.
n.d.
1---5
12--12--5
12--5
-2--5
1---5
12--5
1 - 3412--5
+
+
+
+
–
+
+
–
+
–
+
–
+
–
+
+
+
+
–
–
–
–
–
–
–
–
–
–
The percentage of identical amino acid residues was calculated for the complete protein sequences (not available for AtSTP5). Calculations
were performed using the program bestfit of the Genetics Computer Group, Inc. (gap creation penalty = 12; gap extension penalty = 4).
AtSTP genes are interrupted by 2 or 3 introns that are located at conserved positions (position 1, 2, 3, 4 or 5). The intron positions are
indicated.
well. Nevertheless, this experiment showed that at least 11
independent genes for putative monosaccharide transporters exist in Arabidopsis. The corresponding genes of
these new putative monosaccharide transporters were
named AtSTP5 to AtSTP11. In addition, data libraries were
screened with these AtSTP sequences and the results were
included in Table 1. For eight of the 11 AtSTP clones EST
and/or genomic clones were identified. Only for one of our
PCR clones were (AtSTP5, AtSTP9 and AtSTP10) no
matching sequences found in any of these data libraries.
However, three homologous Arabidopsis genes were identified in these data base searches that had not been picked
up during the PCR analyses. These genes were named
AtSTP12 to AtSTP14 and the details of these genes are
included in Table 1.
Our analyses show that Arabidopsis possesses a family of
at least 14 homologous AtSTP genes. So far, four of these
genes have been functionally characterized as monosaccharide transporters by heterologous expression in fission
yeast or baker’s yeast. The high degree of identity between
the individual members of this gene family (Table 1) suggests that the other members of this family may also encode
monosaccharide transporters.
DISCUSSION
AtSTP3 encodes a monosaccharide transporter
with unusual properties
In the presented paper the AtSTP3 gene is characterized as
a new member of the AtSTP monosaccharide transporter
family. As with previously analysed monosaccharide transporters from higher plants, AtSTP3 catalyzes the energydependent transport of hexoses and pentoses and
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
transports fructose only at very low rates. However, with its
high Km value for glucose (2 mm) AtSTP3 represents the
first low affinity monosaccharide transporter and with its
localization in green leaves AtSTP3 is the first transporter
gene that is not expressed in a classical sink tissues. The
physiological role of a green leaf specific monosaccharide
transporter may be the retrieval of sugars being lost from
the cytoplasm by passive leakage. A function of AtSTP3 in
cell-to-cell transport of monosaccharides between different
cells of the mesophyll is unlikely. These cells have been
shown in several plant species to be symlastically connected
by plasmodesmata with size exclusion limits of about 1 kDa
(Tucker 1982; Goodwin 1983; Wolf et al. 1989).
Besides AtSTP4, AtSTP3 represents the second stressregulated monosaccharide transporter gene of Arabidopsis.
In contrast to AtSTP4 that exhibits a rapid wound response
with maximal expression levels being reached 60 to 120 min
after wounding (Truernit et al. 1996), the induction of
AtSTP3 expression is slow and continues over a much
longer time (Fig. 6) excluding a monosaccharide import
function of AtSTP3 during the immediate and early wound
response reactions. Plant wound responses can obviously be
divided into rapid and slow responses and similar differences in the induction time course have previously been
described for several wound induced genes of hydroxyproline-rich glycoproteins of Phaseolus vulgaris (Sauer et al.
1990a).
Our results confirm earlier studies describing an induction of 3-O-methylglucose uptake in wounded squash
tissue (Hancock 1969, 1970) and may be interpreted as a
wound-induced sink formation.The increased expression of
AtSTP3 may thus reflect the physiological necessity to
support wounded tissue with additional carbohydrates for
additional metabolic tasks. This is supported by the wound-
182 M. Büttner et al.
induced activity of an extracellular invertase that has been
described in carrots (Sturm & Chrispeels 1990). The coordinated activities of monosaccharide transporters and cell
wall-bound invertases are regarded as crucial for the supply
of carbohydrates via an apoplastic pathway (Ehneß &
Roitsch 1997) and expression of a yeast-derived invertase
in the apoplast of tobacco plants caused indeed an accumulation of hexoses in leaf tissue (Heineke et al. 1994).
Alternatively, AtSTP3 that is not expressed in sink tissues
of unwounded Arabidopsis plants may function in the
retrieval of monosaccharides (glucose, galactose, xylose,
etc.) that are released during cell damage and cell wall
degradation.
The present data show also that the formation of new
sinks, e.g. by wounding, is accompanied by the enhanced
expression of monosaccharide transporter genes, such as
AtSTP3 and AtSTP4. Phloem loading is not enhanced and
the mRNA levels of the companion cell-specific AtSUC2
sucrose transporter (Stadler & Sauer 1996) are not affected
by this treatment (Fig. 6b). This suggests that the flow of
assimilates towards different sinks is modulated primarily
by the increased import of assimilates into these sinks but
not by increased phloem loading.
the same BAC clone derived from chromosome IV. Moreover, different AtSTP genes located on the same chromosome were identified on non-overlapping BAC clones and
the adjacent nucleotide sequences are clearly different.This
suggests that AtSTP1 to AtSTP14 represent individual nonallelic chromosomal loci.
Typically AtSTP genomic sequences are interrupted by
two or three introns that can be located at five different
positions. In most of the genes these positions are precisely
conserved (positions 1, 2 and 5). Only the positions determined for two of the three introns in AtSTP13 (positions 3
and 4) are unique suggesting that AtSTP13 may represent
an evolutionary old member of the Arabidopsis AtSTP
family. This is supported by the phylogenetic tree presented
in Fig. 7. It was generated for those AtSTPs, of which complete protein sequences are available. The sequence of the
yeast ScGAL2 galactose permease (Nehlin, Carlberg &
Ronne 1989) was used as outgroup. Obviously, the AtSTP
family falls in two groups of more closely related proteins
(AtSTP2, AtSTP6, and AtSTP8 versus the other AtSTP
transporters). AtSTP7, AtSTP13 and AtSTP14 cannot be
clearly asigned to one of the two groups and especially
AtSTP13 seems to represent a very basic member of the
AtSTP family.
Arabidopsis has a family of at least 14
(putative) monosaccharide transporters
Using oligonucleotide primers binding to conserved
sequences in the so far characterized Arabidopsis monosaccharide transporter cDNAs (Weig et al. 1994) we were
able to isolate fragments of seven new genes, AtSTP5 to
AtSTP11. Data bank searches with these fragments identified genomic and/or EST sequences for AtSTP1, AtSTP2,
AtSTP3, AtSTP4, AtSTP6, AtSTP7, AtSTP8 and AtSTP11
and for three genes that were not detected during our PCR
analyses, AtSTP12 to AtSTP14. Although not directly
proven by transport analyses with each of the encoded
proteins, it is likely that all of these genes code for monosaccharide transporters. Together with sequences from bacteria, mammals, fungi, lower plants and other higher plants
AtSTP1 to AtSTP14 form a subcluster within the major
facilitator superfamily (Marger & Saier 1993; Büttner &
Sauer, 2000). All of the functionally characterized proteins of this subcluster turned out to be monosaccharide
transporters.
The currently available informations on the Arabidopsis
AtSTP gene family are summarized in Table 1. Ten of the
14 genes have been localized to a specific chromosome. The
proteins encoded by this gene family are between 499
and 542 amino acids long and share between 50 and 80%
identical amino acids. More detailed sequence comparisons
revealed 85·1 or 87·4% identity also between AtSTP9 and
AtSTP10 or between AtSTP6 and AtSTP8. The possibility
that some of the AtSTP genes represent different alleles of
the same chromosomal locus can most likely be excluded.
AtSTP4 and AtSTP10 represent directly adjacent genes on
chromosome V and AtSTP8 and AtSTP13 are located on
Figure 7. Phylogenetic tree of AtSTP protein sequences.
Bootstrap values shown at internal nodes indicate the percentage
of the occurence of these nodes in 1000 replicates (first value;
neighbour joining) or 100 replicates (second value; maximum
parsimony) of the data set. The sequence of the Saccharomyces
cerevisiae galactose permease (Gal2p) was used as outgroup
(accession number: g171553). For AtSTP accession numbers see
Table 1.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184
A family of monosaccharide transporters in Arabidopsis 183
Several of the AtSTP family members have been characterized with respect to their site of gene expression and
with respect to their kinetic properties. They differ only
marginally in their substrate specificities and all of them are
energy-dependent H+-symporters. As shown in this paper
they can differ in their substrate affinities by a factor of up
to 100. Most importantly, however, there is no overlap in
the site and time of gene expression of the so far characterized AtSTP genes suggesting that also the other
members of this gene family may be expressed at distinct
sites for a certain time or in response to a specific environmental or developmental signal.
ACKNOWLEDGMENTS
We thank Carola Hümmer for excellent technical help.
This work was supported by grants to N.S. from the
Deutsche Forschungsgemeinschaft (Sa 382/7-1) and from
the European Cummunity (PL 960583).
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Received 23 June 1999; received in revised form 23 September 1999;
accepted for publication 23 September 1999
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 175–184