SUT2, a Putative Sucrose Sensor in Sieve Elements
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00-0048The Plant Cell, Vol. 12, 1153–1164, July 2000,
www.plantcell.org © 2000 American Society of Plant
Physiologists
SUT2, a Putative Sucrose Sensor in Sieve Elements
Laurence Barker,
b
Department of Plant Biology, Royal Veterinary and Agricultural
University, KVL, Thorvaldsensvej 40, DK-1871 Frederiksberg C,
Copenhagen, Denmark
c
Max Planck Institut für Züchtungsforschung, Carl von Linné Weg 10,
50829 Cologne, Germany
In leaves, sucrose uptake kinetics involve high- and low-affinity
components. A family of low- and high-affinity sucrose transporters
(SUT) was identified. SUT1 serves as a high-affinity transporter
essential for phloem loading and long-dis- tance transport in
solanaceous species. SUT4 is a low-affinity transporter with an
expression pattern overlapping that of SUT1. Both SUT1 and SUT4
localize to enucleate sieve elements of tomato. New sucrose
transporter–like proteins, named SUT2, from tomato and Arabidopsis
contain extended cytoplasmic domains, thus structurally resembling
the yeast sugar sensors SNF3 and RGT2. Features common to these
sensors are low codon bias, environment of the start
codon, low expression, and lack of detectable transport activity.
In contrast to
LeSUT1
SUT2
is more highly expressed in sink than in source leaves and is
inducible by su- crose. LeSUT2 protein colocalizes with the low-
and high-affinity sucrose transporters in sieve elements of tomato
pet- ioles, indicating that multiple SUT mRNAs or proteins travel
from companion cells to enucleate sieve elements. The
SUT2
gene maps on chromosome V of potato and is linked to a major
quantitative trait locus for tuber starch content and yield. Thus,
the putative sugar sensor identified colocalizes with two other
sucrose transporters, differs from them in kinetic properties, and
potentially regulates the relative activity of low- and
high-affinity sucrose transport into sieve elements.
INTRODUCTION
Sucrose, the major product of photosynthesis in mature leaves, is
loaded into the vascular tissue for translocation to heterotrophic
tissues to support their growth. In solana- ceous plants, SUT1 is
essential for phloem loading into sieve elements (Riesmeier et al.,
1994; Kühn et al., 1996; Bürkle et al., 1998). SUT1 serves as a
high-affinity trans- porter for sucrose (
K
m
z
1 mM; Riesmeier et al., 1993), whereas SUT4, with a
K
m
of
z
11 mM, is a low-affinity su- crose transporter (Weise et al.,
2000). Both proteins colocal- ize in sieve elements (Kühn et al.,
1997; Weise et al., 2000). Localization of SUT1 protein in sieve
elements and SUT1 mRNA at the orifices of plasmodesmata
interconnecting companion cells and sieve elements, together with
the high turnover of both SUT1 mRNA and protein, indicate that
traf- ficking of mRNA or protein occurs from companion cells into
enucleate sieve elements by way of plasmodesmata (Kühn et al.,
1997).
Sugar transport is highly regulated, and sucrose-specific
signaling pathways are involved in controlling transport ac- tivity
(Chiou and Bush, 1998), potentially by using protein
phosphorylation (Roblin et al., 1998). Overexpression of pyruvate
decarboxylase in potato leads to a 10-fold in- crease in sugar
export, demonstrating the capacity to regu- late sugar export from
leaves within a wide dynamic range (Tadege et al., 1998). This
poses the question of how regula- tion is coordinated between sieve
elements that contain the transporters and companion cells in which
transcription takes place (Lalonde et al., 1999).
Yeast, for which the study of transporter regulation is much
further advanced, may provide hints as to the general mechanism of
regulation of sugar transport. Complex regula- tory networks
control uptake of nutrients in response to intracellular needs and
a rapidly changing external environ- ment. For metabolization of
sugars, yeast has developed a two-step regulatory system to ensure
coordination between the external availability of sugars and the
enzymatic machin- ery inside cells: (1) the extracellular
concentration of sugars is sensed, and sugar transport activity is
regulated accord- ingly; and (2) sugar transport activity
determines the flux of sugars into the cell, subsequently
generating intracellular sig- nals for adapting metabolism (Lalonde
et al., 1999).
1
1154 The Plant Cell
The 18 yeast monosaccharide transporters found thus far span a wide
range of affinities, and thus, depending on the external sugar
availability, the kinetic parameters of uptake can be adapted
accordingly, for instance, by changing from low to high affinity if
sugar availability drops (Lalonde et al., 1999; Wieczorke et al.,
1999). HXT2 and HXT7, for example, which serve as high-affinity
glucose transporters, are in- duced by low concentrations of
glucose but are repressed at high concentrations, whereas the
low-affinity transporter HXT1 is induced by high concentrations of
glucose (Özcan and Johnston, 1995; Boles and Hollenberg, 1997).
Conse- quently, the sensors for extracellular glucose must respond
not only to the kind of carbon source but also to its concen-
tration.
Glucose is sensed by SNF3 and RGT2, which trigger the induction of
hexose transporter genes based on the glucose concentration (Liang
and Gaber, 1996; Özcan et al., 1996b). These two proteins resemble
glucose transporters but, in addition, possess unusually long
C-terminal domains that reside in the cytoplasm (Özcan et al.,
1996a). The large C-ter- minal extension of SNF3 (303 amino acids)
contains two nearly identical repeats of 25 amino acids. One of
these re- peats is also present in RGT2. The C-terminal domains of
SNF3 and RGT2 mediate the signaling function of the pro- teins
(Özcan et al., 1998; Vagnoli et al., 1998). Deletion of these
extensions leads to a loss of sensor function. Attach- ment of the
SNF3 C terminus to glucose transporters HXT1 and HXT2 confers the
ability to signal glucose availability. Apparently, SNF3 is a
sensor of low glucose concentrations that mainly regulates
expression of high-affinity glucose trans- porters, whereas RGT2 is
responsible for sensing high glu- cose concentrations and
regulating expression of low- affinity transporters. Consequently,
induction of the high- affinity transporter HXT2 by low
concentrations of glucose is absent in
snf3
mutants, and induction of the low-affinity transporter HXT1 by high
concentrations of glucose is strongly reduced in
rgt2
mutants. In addition, SNF3 is re- quired for repression of the
high-affinity transporters HXT2, -6, and -7 at high glucose
concentrations (Liang and Gaber, 1996; Vagnoli et al., 1998).
Despite the high homology of SNF3 and RGT2 to glucose permeases,
neither appears to be able to mediate substantial glucose transport
(Liang and Gaber, 1996; Özcan et al., 1998).
Little is known about sugar sensing and signaling with respect to
transport of sucrose and glucose in plants (Hellmann et al., 2000).
Yeast thus may serve as a model to improve our understanding of the
role of sugar transporters in sugar sensing. The existence of
plasma membrane– bound sensing pathways has been suggested from
physio- logic analyses using nonmetabolizable glucose analogs
(Martin et al., 1997; Roitsch, 1999). Furthermore, a genetic
approach has led to identification of sugar-regulated signal- ing
cascades that modulate sugar uptake (Hellmann et al., 2000).
To investigate whether plants contain multiple sucrose transporters
present in sieve elements and to identify puta-
tive sugar sensors, we initiated a search for additional su- crose
transporter–like genes. A new sucrose transporter– like protein,
SUT2, was identified that colocalizes with low- and high-affinity
sucrose transporters in enucleate sieve ele- ments of tomato and
shares features with yeast sugar sen- sors. Most importantly,
SUT2
is a candidate gene for a major quantitative trait locus (QTL) for
tuber starch and yield in potato, the genome of which is colinear
with that of to- mato (Gebhardt et al., 1991).
RESULTS
Two novel
homologs were identified from tomato by low-stringency
hybridization and reverse transcription–poly- merase chain reaction
(PCR).
LeSUT1
StSUT1
from potato (94% similarity of amino acid se- quences) and appears
to be the ortholog of
StSUT1
LeSUT2,
with a cDNA of 2144 bp, encodes a 65-kD protein with unique
features not reported previously for SUT homologs. SUT2 has an
N-terminal do- main
z
30 amino acids longer than the sucrose transporters already
identified (see Lalonde et al., 1999), and its ex- tended central
cytoplasmic loop is
z
50 amino acids longer (Figure 1A). According to high-stringency DNA
gel blot anal- ysis,
LeSUT2
does not have a closely related paralog in the tomato genome
(Figure 2B). Screening bacterial artificial chromosome and
expressed sequence tag databases led to the identification of a
putative ortholog of
LeSUT2
AtSUT2
, that contains the same two extended do- mains (Figure 1B). The
SUT2s are 82% similar (68% identical), and the genes are
structurally related, that is, the intron–exon borders and the
number of introns are con- served (Figure 2A). Besides the domains
that are conserved in all sucrose transporter–like proteins, LeSUT2
and AtSUT2 share highly conserved sequence motifs within the ex-
tended central loop, suggesting a functional similarity be- tween
AtSUT2 and LeSUT2 as well (Figures 1B and 1C). Similar to the yeast
hexose sensors, both SUT2 proteins are characterized by low codon
bias and low homology with Kozak consensus (Kozak, 1996),
indicating a low translation efficiency (Iraqui et al., 1999; J.M.
Ward, unpublished re- sults).
Expression of
LeSUT2
AtSUT2
) for func- tional characterization (Rentsch et al., 1995). LeSUT1
com- plemented the yeast mutant SUSY7/ura3 (Riesmeier et al.,
1992), consistent with being the ortholog of StSUT1 (Figure 3A),
whereas LeSUT2 and AtSUT2 were unable to comple-
Putative Sucrose Sensor 1155
9
LeSUT2
were removed and a dif- ferent vector (YEPlac112A1NE) was used
(Riesmeier et al., 1992). For all constructs, a variety of
complementation con- ditions was tested, including sucrose
concentrations be- tween 0.5 and 2% and the pH of the medium
ranging from 4.0 to 7.0.
To verify expression of LeSUT2, we raised polyclonal anti- bodies
against synthetic peptides of LeSUT2 and used them for protein gel
blot analysis. Specificity of the antisera was verified on membrane
proteins isolated from yeast cells ex- pressing LeSUT2. LeSUT2
migrates as a larger protein than LeSUT1, as their sequences would
predict, and appears as a doublet at
z
65/67 kD, possibly because of post-trans- lational modifications in
yeast (Figure 3B). No signals were detected in yeast transformed
with the empty vector. Anti- bodies raised against StSUT1 located
LeSUT1 in plasma membranes that had been purified from tomato
leaves and showed it to migrate at an apparent molecular mass of 46
kD (Figure 3B). Sequences chosen for antisera produc- tion were
unique. Lack of cross-reactivity with known LeSUTs was confirmed by
immunolocalization in yeast (Fig- ure 4).
Expression of Sucrose Transporter Genes in Tomato
RNA gel blot analysis was performed to investigate organ- specific
expression of
LeSUT1
and
LeSUT2
LeSUT1
was most highly expressed in source leaves, similar to its ortho-
log in potato (Riesmeier et al., 1993), with lower expression
Figure 1.
Structural comparison of SUT1 and SUT2.
Hydrophobicity plots (Kyte and Doolittle, 1982) (window of 11 amino
acids) of LeSUT1 and LeSUT2 were overlayed. Both contain 12
putative transmembrane domains (labeled with roman numerals I to
XII), but SUT2 contains several hydrophilic domains not present in
SUT1.
(B)
Predicted topology of LeSUT2. All amino acids conserved in both
SUT2 orthologs are shown in black. The central loop region contains
two highly conserved sequence motifs: central conserved box (CCB) 1
and 2, which are present only in SUT2 orthologs (under-
lined).
(C)
Homology of sucrose transporters. The tree is based on maxi- mum
parsimony analysis (Swofford, 1998) for protein sequences of
aligned sucrose transporters from tobacco (NtSUT1 [Bürkle et al.,
1998] and NtSUT3 [Lemoine et al., 1999]), potato (StSUT1 [Riesmeier
et al., 1993]), tomato (LeSUT1, LeSUT2, and LeSUT4), Arabidopsis
(AtSUC1, AtSUC2 [Sauer and Stolz, 1994], AtSUT2, and AtSUT4),
and
Schizosaccharomyces pombe
(SpSUT1 [CAB16264]). Percent- age bootstrap values of 1000
replicates are given at each branch point. Branch lengths (drawn in
the horizontal dimension only) are proportional to phylogenetic
distance.
1156 The Plant Cell
SUT2
was low in general but was higher in sink leaves and stems than in
source leaves.
Because sucrose transporter activity has been shown to be regulated
by sugars in sugar beet (Chiou and Bush, 1998), the regulatory
effect of sugars on sucrose transporter expression was investigated
in tomato and potato.
SUT2
was induced in the presence of 100 mM sucrose in source leaves from
potato.
SUT1
expression remained unaltered, whereas patatin (a storage protein
expressed in potato tu- bers) expression was induced primarily by
sucrose and to a lesser extent by glucose (Figure 5B). In tomato,
the sucrose induction of
LeSUT2
was seen in sink leaves but not in source leaves (data not
shown).
Again,
SUT1
Colocalization of SUT1 and SUT2 in Tomato Petioles and Stems
For localization of LeSUT1 and LeSUT2 in tomato, antisera were
affinity purified and used for immunocytochemistry. Both
transporters were localized exclusively in sieve ele- ments, as was
LeSUT4 (Weise et al., 2000). The presence of sieve plates, together
with the absence of nuclei, allowed unambiguous identification of
sieve elements (Figures 6A, 6B, and 6D). LeSUT1 was present in
sieve elements of source-leaf minor veins (Figure 6H), whereas SUT2
was de- tected in the midrib of sink and source leaves but not in
mi- nor veins (Figures 6F
and
6G). All three transporters— LeSUT1, LeSUT2 (Figures 6A and 6B),
and LeSUT4—colo- calized in mature sieve elements of stem and
petiole. Elec-
Figure 2. Genomic Structure of the SUT Loci.
(A) Comparison of the genomic structure of LeSUT2 versus AtSUT2,
showing the high conservation of the number of exons (exons 1–14).
(B) DNA gel blot analysis of the LeSUTs under high stringency.
Genomic DNA from tomato was digested with restriction enzymes,
resolved on a 1% agarose gel (20 mg/lane), and blotted to Hybond
N1. The gel at left shows hybridization with a 32P-labeled 1.3-kb
fragment of LeSUT1. The gel at right shows hybridization with a
32P-labeled LeSUT2 cDNA.
Putative Sucrose Sensor 1157
tron microscopic immunogold labeling clearly showed that LeSUT2 is
present at the plasma membrane (Figure 6J). Pre- immune serum,
purified on protein A–Sepharose and used at the same protein
concentration as LeSUT2, caused weak cytoplasmic background
labeling, although not at plasma membranes (Figure 6K).
A series of control experiments was performed to exclude any bias
from nonspecific binding. This was necessary be- cause of the
preferential binding of immunoglobulins to sieve elements at high
concentrations, which was detect- able after long exposures. No
immunofluorescence was detectable with IgG-enriched purified
preimmune serum (Figure 6E), nor after incubation with the
secondary antibody alone (data not shown). Confirmation that
purified antisera were specific resulted from immunolocalization on
sphero- plasted yeast cells transformed with transporter genes or
an empty expression vector (Figure 4). Localization with a sec- ond
antiserum against different domains of SUT1 and SUT2 further
confirmed results obtained with the first antiserum. Similar
results were obtained when tissue was embedded in acrylic resins
after conventional fixation or when tissue was prepared by
high-pressure freezing (data not shown). Antibody–peptide
competition experiments also were per- formed to exclude
nonspecific antiserum binding. Fluo- rescent signals resulting from
SUT2 detection were strongly reduced or abolished after
preincubating the SUT2 antise- rum with its corresponding peptide
(Figure 6C). However, preincubation with a peptide corresponding to
another epitope of the same transporter or to a control peptide had
no effect on SUT2 signals (Figure 6D).
AtSUT2 Expression in Transgenic Plants
Transgenic Arabidopsis plants expressing the reporter gene
for
b
-glucuronidase (GUS) under control of a 1.2-kb AtSUT2 promoter
fragment showed GUS staining in the vascular tis- sue of the
petiole and in the major and second-order veins of source leaves.
Among 10 hygromycin-resistant transfor- mant lines, nine showed the
same GUS expression. In a few cases, weak staining of third-order
veins was detectable. Staining also was observed in trichomes of
source leaves and in hydathodes (Figure 6I). For the longer 2.2-kb
pro- moter–GUS construct, only two transformants were ana- lyzed,
both showing the same GUS expression pattern as seen for the
shorter construct.
Map Positions of
Genes in Potato
When used as restriction fragment length polymorphism (RFLP)
markers on the potato molecular map, the three cDNA clones each
mapped to a separate locus.
StSUT1
LeSUT4
SUT2
DISCUSSION
The mechanisms for regulation of sucrose transport during loading
and unloading are not fully understood. Sucrose up- take kinetics
determined in plasma membrane vesicles are multiphasic, suggesting
the existence of multiple carrier sys- tems (Lemoine et al., 1996).
Theory also predicts additional carriers in several different cell
types, including sucrose
Figure 3. Expression of LeSUT1 and LeSUT2 in Yeast.
(A) Expression of LeSUT1 and LeSUT2 in SUSY7/ura3. The dish at left
shows that LeSUT1 functionally complements the yeast mutant,
SUSY7/ura3 on sucrose (0.5%), whereas LeSUT2 reduced growth below
that of the empty expression vector. At right, growth on glu- cose
(2%). (B) Protein gel blot analysis of LeSUT1 and LeSUT2. At left
is shown purified tomato source-leaf membranes (50 mg) resolved by
electro- phoresis. LeSUT1 was detected by using an
affinity-purified antise- rum against StSUT1. MM, microsomal
membranes; IM, intracellular membranes; PM, plasma membranes. At
right, LeSUT2 was ex- pressed in SUSY7/ura3, and 50 mg of the total
membrane protein was resolved by electrophoresis. LeSUT2 was
detected as a doublet at 66 kD and was absent in yeast transformed
with the empty ex- pression vector.
1158 The Plant Cell
import and export systems (Lalonde et al., 1999). In addition to
SUT1, three other sucrose transporters have been identi- fied in
solanaceous species: LeSUT4, which appears to play a role in
low-affinity sucrose transport (Weise et al., 2000); NtSUT3, a
carrier involved in pollen nutrition (Lemoine et al., 1999); and
SUT2, a putative sucrose sensor. All sucrose transporters that have
been isolated have a predicted topol- ogy of 12 transmembrane
domains. SUT2 has not only this predicted structure but also two
additional features: ex- tended N-terminal and central loop domains
that average 30 and 50 amino acids longer, respectively, than those
of other SUT family members. According to hydrophobicity analysis
and experimental evidence for PmSUC2 (Stolz et al., 1999), the N
terminus and central loop are localized intracellularly, which
allows these extended domains to interact with pro- teins in the
cytoplasm. Another characteristic of
SUT2
is the low similarity to the Kozak consensus for high rates of
trans- lation initiation (Kozak, 1996) and a low codon bias,
indicat- ing low translatability.
In contrast to other members of the SUT family, SUT2 does not
complement the yeast mutant that is deficient in sucrose uptake,
and its expression in yeast appears to cause toxicity. Even in a
more sensitive selection system, one deficient in hexose uptake,
which thus leads to reduced background growth, SUT2 is still
nonfunctional. Although an apparently full-length SUT2 protein can
be detected by pro- tein gel blot analysis, the data do not exclude
the possibility that nonfunctionality is the result of insufficient
targeting to yeast plasma membranes or that the large central loop
acts as an autoinhibitory domain.
Putative Sensor Function of SUT2
In tomato, SUT2 was localized to sieve elements, in which it
colocalizes with the high- and low-affinity sucrose transport- ers
SUT1 and SUT4, respectively. SUT2 is regulated by su- crose, adding
to other evidence for the sugar regulation of sucrose transporters
at the plasma membrane level (Chiou and Bush, 1998). In general,
SUT2, which shows many fea- tures similar to the yeast sugar
sensors SNF3 and RGT2, may be involved directly in controlling
expression, activity, and turnover of the other two sucrose
transporters SUT1 and SUT4 and thereby may act to control sucrose
fluxes across the plasma membrane of sieve elements.
Interestingly, the hexose transporter family from Arabi- dopsis
also contains several transporter-like members that contain
extended cytosolic loops, which thus represent po- tential
candidates for hexose sensors (Lalonde et al., 1999). An emerging
mechanism found in many organisms for vari- ous substrates is the
presence of transporters or trans- porter-like proteins involved in
sensing and regulating the
Figure 4. Specificity of the LeSUT2 Antiserum Shown by Yeast Im-
munofluorescence.
LeSUT1, LeSUT2, or the empty vector pDR195 was expressed in
SUSY7/ura3, the cells were fixed, and SUT proteins were detected
with affinity-purified antisera. No cross-reactivity of the SUT1
antise- rum on SUT2-expressing yeast or SUT2 antiserum on SUT1-
expressing yeast was detected. No signals were detected on yeast
expressing pDR195.
Figure 5. Expression of SUT2.
(A) Organ-specific expression of LeSUT1 and LeSUT2 (20 mg/lane) in
stringent hybridization conditions. Probes were the full-length
cDNA of LeSUT1 and a 1.3-kb fragment of LeSUT2. (B) Comparison of
SUT1, SUT2, and patatin expression under differ- ent treatments in
potato. SUT2 was specifically induced after treat- ment with
sucrose (100 mM), whereas SUT1 expression remained unchanged. Both
sucrose and to a lesser extent glucose (100 mM) induced patatin
expression. Sorbitol also was used as an osmotic control at a
concentration of 100 mM.
Putative Sucrose Sensor 1159
Figure 6. Immunocytochemical Localization of SUT Proteins.
(A) Immunofluorescent detection of SUT1 in enucleate sieve elements
of tomato stems, colocalized with the aniline blue–stained callose
in the sieve plate. DAPI-stained nuclei are visible in neighboring
phloem cells. (B) SUT2 colocalized with aniline blue–stained sieve
plate callose in enucleate sieve elements of stem. (C) and (D) Loss
of LeSUT2 signal after incubation of SUT2 antiserum with its
corresponding peptide (C). No reduction in signal after incubation
of SUT2 antiserum with a control peptide (D). (E) No signals
detected with preimmune serum; aniline blue–stained sieve plate
callose indicates presence of sieve element. (F) No
immunofluorescence labeling detectable in source-leaf minor veins
of tomato plants treated with anti-LeSUT2 antiserum. (G)
Bright-field view of the same section as in (F), showing minor vein
anatomy. (H) LeSUT1 detectable in source-leaf minor veins on a
serial section as in (F). (I) Source leaf of a transgenic
Arabidopsis plant transformed with the 1.2-kb AtSUT2 promoter–GUS
fusion construct. GUS staining is detect- able in first-, second-,
and third-order veins but not in minor veins. (J) Immunogold
labeling of the sieve element plasma membrane in a petiole
cross-section by using anti-LeSUT2 antibodies. (K) Control treated
with IgG-enriched preimmune serum on a serial section as in (J).
Bar in (A) to (H) 5 40 mm; bar in (J) and (K) 5 500 nm. n, nucleus;
pp, phloem parenchyma cell; se, sieve element; sp, sieve plate; w,
cell wall.
1160 The Plant Cell
activity of carriers according to substrate requirement, such as
the bacterial iron/citrate transporter/sensor FecA, the yeast amino
acid sensor SSY1, and the putative plant metal sen- sor EIN2 (Boles
and Hollenberg, 1997; Alonso et al., 1999; Braun and Killmann,
1999; Iraqui et al., 1999).
High conservation of central sequence motifs within the extended
cytoplasmic loops of SUT2 proteins (CCB1 and CCB2) suggests an
important role for SUT2 function. Ex- tended cytoplasmic domains
could represent domains for effector binding or signal
transduction. For example, the CCB2 domain contains a serine
residue at a highly con- served position, which is predicted to be
the target for regu-
lation by phosphorylation by a serine/threonine kinase. Sucrose
transport already has been described to be regu- lated by
phosphorylation (Roblin et al., 1998).
SUT2 Trafficking into Sieve Elements
The present study of SUT2 localization together with a par- allel
analysis of SUT4 has shown that three SUT proteins, falling into
three clusters, colocalize in sieve elements (Fig- ure 1C; Weise et
al., 2000). SUT1 is likely to be transported from the companion
cell to the sieve element, probably in the form of mRNA, given that
both its mRNA and protein turn over rapidly (Kühn et al., 1997).
Previous studies show- ing that SUT1 mRNA localizes to the orifices
of plasmodes- mata both in companion cells and in sieve elements
lead to the suggestion that all three SUTs use similar trafficking
mechanisms for their mRNA. The mechanism may involve
receptor-mediated transport of RNA, similar to that occur- ring in
Drosophila oocytes (St. Johnston, 1995), possibly involving a
CmPP16 homolog that has been shown to in- crease SUT1 mRNA mobility
between mesophyll cells (Xoconostle-Cázares et al., 1999). Although
the structural requirements of the mRNA that allow for
plasmodesmatal movement have not been identified, investigation
into the in- volvement of cis elements or a common RNA structure in
the three SUT genes may help reveal such a mechanism.
SUT2 Is Expressed in the Transport but Not in the Loading
Phloem
Expression of the AtSUT2 promoter reporter gene fusion was found in
the vascular tissue of leaf major veins in trichomes and in
hydathodes but not in minor veins, the places of phloem loading.
These data are consistent with the immu- nolocalization data of
LeSUT2. Different parts of the phloem were classified according to
their different functions into the collection phloem, the transport
phloem, and the delivery phloem (van Bel, 1996). Anatomic
characteristics reflect the different tasks of the phloem (i.e.,
the size relationship be- tween the companion cells and the sieve
elements: sieve ele- ments of the transport phloem, to which a
retrieval function was assigned, are bigger than the neighboring
companion cells). Thus, differences in the localization of SUT1,
which is strongly expressed in source-leaf minor veins (Riesmeier
et al., 1993; Kühn et al., 1997), and SUT2, which is localized in
the transport phloem, indicate a strikingly different function of
the two sucrose transporter–like proteins.
Linkage of SUT Genes to QTLs for Tuber Starch Content and Tuber
Yield
Transport of sucrose from source leaves to sink tubers is essential
for starch accumulation in developing tubers.
Figure 7. Mapping of SUT Loci.
cDNA clones StSUT1, LeSUT4, and LeSUT2 were used as RFLP markers in
mapping population BC9162 and identified SUT1 (StSUT1) on linkage
group XI, SUT4 (LeSUT4) on linkage group IV, and SUT2 (LeSUT2) on
linkage group V. Centimorgan distances be- tween SUT loci and
anchor RFLP markers (Gebhardt et al., 1999) are indicated to the
left of the linkage groups. GP and CP markers are anonymous potato
genomic and cDNA fragments, respectively. Small letters in
parentheses indicate that more than one locus was identified by the
marker probe. Functional gene markers (Gebhardt et al., 1999) are
as follows: BE, branching enzyme; MBF, mas bind- ing factor; StPto,
Solanum tuberosum homolog of tomato resistance gene Pto; UGPase,
UDP–glucose pyrophosphorylase. Positions of QTLs for tuber starch
content [ts(i), ts(k), ts(n), ts(a)] and tuber yield [yi(a)], shown
to the left of the linkage groups, are derived from Schäfer-Pregl
et al. (1998), based on anchor RFLP markers with known genetic
distance to QTL and SUT loci.
Putative Sucrose Sensor 1161
Genetic variability of the genes controlling sucrose transport
therefore may have a quantitative effect on the starch con- tent of
mature tubers. QTLs for tuber starch content and tu- ber yield have
been positioned on the potato RFLP map. QTLs for tuber yield and
tuber starch content are linked, suggesting that at least some
factors controlling tuber starch content have pleiotropic effects
on tuber yield (Schäfer-Pregl et al., 1998). Given their essential
physiologic role, genes controlling sucrose transport are
candidates for QTLs controlling tuber starch content. If alleles at
one or more transporter or sensor loci indeed are responsible for a
QTL effect on tuber starch content, tight genetic linkage will be
observed between the QTL and the candidate gene lo- cus. Therefore,
the positions of SUT loci were compared with the positions of QTL
for starch content and tuber yield.
Three unlinked SUT loci were identified in the potato mo- lecular
map: SUT4 on linkage group IV, SUT1 on linkage group XI, and SUT2
on linkage group V. On linkage group IV, QTLs for tuber starch
content are located in the interval flanked by RFLP loci GP180(a)
and GP261(a) and are closely linked to the branching enzyme locus
BE (Schäfer-Pregl et al., 1998). SUT4, however, maps in between the
two QTLs (Figure 7). Similarly, a minor QTL for tuber starch
content is linked to marker loci GP125 and UGPase (UDP–glucose py-
rophosphorylase) on linkage group XI (Schäfer-Pregl et al., 1998),
whereas SUT1 occupies a more proximal position on the same linkage
group (Figure 7). On linkage group V, a major QTL for tuber starch
content and tuber yield is linked to markers GP179 and GP291(a)
(Schäfer-Pregl et al., 1998). The SUT2 locus is tightly linked to
the same markers (Figure 7) and therefore to this QTL. This linkage
suggests that (1) SUT2 alleles may play an important role in
controlling tuber starch content in the field and (2) SUT2 alleles
may have pleiotropic effects on tuber starch content and tuber
yield. Further work is required to confirm this important role, be-
cause linkage analysis cannot exclude the possibility that the gene
controlling the QTL is linked, but is functionally un- related to,
the candidate gene locus SUT2.
METHODS
LeSUT Genes and Expression Analysis
Screening a flower cDNA library from Lycopersicon esculentum cv
UC82b with an NtSUT3 genomic fragment (Lemoine et al., 1999) un-
der low stringency led to the isolation of 15 LeSUT1 cDNA clones.
The partial genomic sequence of LeSUT2 was isolated by screening a
genomic library from L. esculentum cv VFN8 in EMBL-3 (Clontech,
Palo Alto, CA). By hybridization with StSUT1 under nonstringent
con- ditions, 11 positive l phages were obtained. One of the more
weakly hybridizing l phages (17/2) represented LeSUT2. A 2.1-kb
EcoRI fragment was used to identify the LeSUT2 cDNA from a leaf
cDNA li- brary from L. esculentum VC82b, in l-ZAPXR
(Stratagene).
The partial genomic LeSUT2 clone was characterized further by using
a 2.1-kb XhoI fragment, overlapping the LeSUT2 cDNA from
positions 274 to 604 upstream of the ATG, and a 4-kb genomic XhoI
fragment, containing sequences from position 802 to the end of the
LeSUT2 cDNA. The gap in the genomic sequence between the two
fragments was filled by polymerase chain reaction (PCR) of the DNA
of the l-phage isolate 17/2 with the use of appropriate
primers.
RNA was isolated from different organs of greenhouse grown L. es-
culentum Moneymaker, as described (Schwacke et al., 1999). RNA gel
blots were hybridized in 50% formamide at 428C and washed with 0.2
3 SSC/0.1% SDS at 658C (1 3 SSC is 0.15 M NaCl and 0.015 M sodium
citrate). For DNA gel blot analysis, genomic DNA from L. esculentum
was digested with restriction enzymes, resolved on 1% agarose gels,
and blotted to Hybond N1 (Amersham Pharmacia Biotech). Hybridiza-
tion was performed with 32P-labeled probes under high stringency,
fol- lowed by washing at 658C in 0.2 3 SSC/0.1% SDS.
The LeSUT2 sequence was deposited in GenBank under the ac- cession
number AF166498.
AtSUT2 Analysis
The cDNA of AtSUT2 was cloned by reverse transcription–PCR from
Arabidopsis thaliana Columbia (Col-0) ecotype leaves. After reverse
transcription of 5 mg of total leaf RNA, PCR was performed by using
specific primers with the cloning sites EcoRI (f) and XhoI (r), and
the product was cloned into the yeast expression vector pDR196. The
genomic sequence of AtSUT2 is in GenBank under the accession number
AC004138.
The AtSUT2 promoter fragment was isolated by PCR on A. thaliana
Col-0 ecotype genomic DNA by using Pfu-Polymerase (Stratagene). A
transcriptional fusion was generated by cloning 2.2- and 1.2-kb
promoter fragments into pGPTV-HPT (Becker et al., 1992).
Arabidopsis was transformed by vacuum infiltration of trans- formed
Agrobacterium GV2260 in 0.53 Murashige and Skoog me- dium
(Murashige and Skoog, 1962) containing 5% sucrose, 0.005% Silwet,
and 0.044 mM benzylaminopurine (Clough and Bent, 1998). Selection
was performed on Murashige and Skoog medium contain- ing 50 mg/L
hygromycin. Plant material was infiltrated under vacuum with 2 mM
5-bromo-4-chloro-3-indolyl-glucuronide, 50 mM sodium phosphate
buffer, pH 7.2, and 0.5% Triton X-100 and was incubated at 378C for
12 to 16 hr (Jefferson et al., 1987). Destaining was per- formed in
70% ethanol.
Sugar Induction
Plants were grown in the greenhouse with a 16-hr-light/8-hr-dark
cy- cle, and leaves were harvested from 3-month-old plants.
Petioles of detached leaves were re-cut while submerged in water,
EDTA (2.5 mM) was added to inhibit callose formation, and the cut
petioles were transferred to solutions containing 100 mM sugar,
where they were kept for 24 hr under greenhouse conditions. All
experiments were repeated independently.
Yeast Strain Construction
To construct SUSY7/ura3, we digested Yep24 (Biolabs, Schwal- bach,
Germany) with NcoI and ApaI (which deleted an internal 173- bp
fragment of the URA3 gene), and then Yep24 was treated with T4
polymerase and blunt-ligated, which yielded pDura3 (W.N. Fischer
and W.B. Frommer, unpublished results). The truncated Dura3
gene
1162 The Plant Cell
was excised with EcoRI and SmaI, and the linearized Dura3 fragment
was used for transformation of SUSY7 (Riesmeier et al., 1992). The
Dura3 mutants were selected on 5-fluoroorotic acid. Deletion of the
internal URA3 fragment was controlled by PCR (data not
shown).
Membrane Isolation and Protein Gel Blot Analysis
Yeast cells were grown in minimal medium (200 mL) to an OD600 of
z1.1. Cells were harvested at 1000g, washed in distilled water, and
then washed in 25 mM Tris-HCl, pH 7.5, containing 5 mM EDTA. Cells
were resuspended in 100 mL of the same buffer. In a small glass
tube, 1 g of glass beads (0.5 mm in diameter) was added, mixed with
3 mL of protease inhibitor mix (0.1 M phenylmethylsulfonyl fluoride
and 0.25 M p-aminobenzamidine in DMSO), and vortex-mixed four times
for 30 sec each. The cell homogenate was centrifuged for 5 min at
1000g, and the membranes were pelleted from the superna- tant by
ultracentrifugation at 100,000g for 60 min. The sediment was
resuspended in 480 mL of a solution of 100 mM sodium phosphate, pH
8.0, 50 mM NaCl, 2.5% b-D-dodecylmaltoside containing 1:100 (v/v)
protease inhibitor mix, and the membranes were solubilized for 5
min on ice. Plant plasma membranes were purified as described
(Larsson, 1985). SDS-PAGE and protein gel blotting have been de-
scribed previously (Lemoine et al., 1996). Immunodetection was by
electrochemiluminescence (Amersham).
Immunocytochemistry
Rabbits were immunized with synthetic peptides coupled to keyhole
limpet hemocyanin corresponding to the N terminus or central loop
of LeSUT2 (N terminus: MDAVSIRVPYKNLKC; central loop: PKN-
EEQRPDKDQGDS). Affinity purification was performed as described
(Lemoine et al., 1996). Preimmune serum was purified as above, ex-
cept that protein A–Sepharose (Bio-Rad) was used instead of pep-
tide affinity. Antiserum for detection of LeSUT1 prepared against
StSUT1 cross-reacts with tomato (Kühn et al., 1997).
Yeast cells prepared for immunolabeling were grown to OD600
z0.75 and fixed in 3.7% formaldehyde for 15 min at 288C. Cultures
(2 mL) were harvested and resuspended for 30 min at 288C in 1 mL of
0.1 M potassium phosphate, pH 6.4, containing 3.7% formaldehyde.
Cells were washed three times in 0.1 M potassium phosphate, pH 6.4,
and once in 1.2 M sorbitol in 0.1 M phosphate citrate buffer, pH
5.9. Cells were incubated in 200 mL of 1.2 M sorbitol/phosphate ci-
trate with 7 mL of zymolase 100T (ICN Biomedicals Research,
Eschwege, Germany) (3 mg/mL in 10% glucose) for 60 min at 288C,
which was followed by washing in 1.2 M sorbitol/phosphate citrate,
resuspen- sion in 300 mL of 1% BSA in PBS (100 mM sodium phosphate,
pH 7.5, and 100 mM NaCl), and incubated overnight at 48C with
primary antibody on an orbital shaker. After three washes in
BSA–PBS, Cy3- coupled secondary antibody diluted 1:100 was added in
BSA–PBS, and the mixture was incubated for 1 hr on an orbital
shaker in dark- ness. After four washes in BSA–PBS, cells were
analyzed by confo- cal laser scanning microscopy TCP-SP (Leica,
Bensheim, Germany).
Fluorescent immunodetection of LeSUTs was performed with
modifications according to Kühn et al. (1997). Hand-cut fragments
(1 mm2) from tomato leaves, stems, and petioles were fixed
overnight under light vacuum in MOPS buffer (50 mM MOPS/NaOH, pH
6.9, 5 mM EGTA, and 2 mM MgCl2) containing 0.1% glutaraldehyde and
6% formaldehyde. After three washes with MOPS buffer on ice, frag-
ments were dehydrated in an ethanol series. After overnight
incuba-
tion in an equal volume solution of ethanol and methacrylate (75%
[v/v] butylmethacrylate, 25% [v/v] methylmethacrylate, 0.5%
benzoine ethyl ether, and 10 mM DTT), the material was embedded in
100% methacrylate. Polymerization took place overnight under UV
light (365 nm) at 48C. Semithin sections (1 mm thick) were mounted
on Histobond slides (Camon, Weisbaden, Germany) and dried at 508C.
To remove the methacrylate, we incubated the slides for 30 sec in
acetone; they were then rehydrated by an ethanol series and blocked
for 1 hr with 2% BSA in PBS. After overnight incubation with
affinity- purified antibodies, the slides were washed twice in PBS
containing 0.1% Tween (PBS-T) and once with PBS, followed by 1 hr
incubation with anti-rabbit IgG–fluorescein isothiocyanate
conjugate. After three sequential washes with PBS-T, PBS, and
distilled water, photo- graphs were taken with a fluorescence
microscope (Axiophot; Zeiss) using fluorescein isothiocyanate
filters. For double-staining of trans- porters and nuclei with
49,6-diamidino-2-phenylindole (DAPI), sec- tions were treated as
described above. After three washes, slides were incubated for 1 hr
in 0.2 mg/mL DAPI. DAPI fluorescence was detected at 365 nm. For
double staining of transporters and sieve plate callose with
aniline blue, slides were incubated for 10 min in 0.05% aniline
blue in 50 mM sodium phosphate, pH 7.2. Aniline blue fluorescence
was detected at 365 nm. For antibody–peptide competi- tion
experiments, affinity-purified antiserum was incubated, before im-
munolocalization, for z16 hr at 48C, either with its corresponding
peptide or with a peptide mimicking another epitope of the same
trans- porter or control peptide (CAFGDPIDSKLSR), at 103 molar
excess.
For electron microscopic immunogold staining, petiole material of
tomato plants was fixed under vacuum for 12 hr with 4% formalde-
hyde/0.1% glutaraldehyde in 100 mM Pipes, pH 7.2. After rinsing in
100 mM Pipes, tissue was dehydrated in ethanol and embedded in
acrylic resins (Unicryl; Plano, Wetzlas, Germany; or LR White; Lon-
don Resin Co., Berkshire, UK). Polymerization took place at 608C
over 2 days. Ultrathin sections (100 nm) were stretched with
chloro- form and mounted on gold grids (Gilder-Grids 400 Hex;
Plano), after which the grids were incubated for 10 min in 0.1 M
HCl and washed with distilled water. After blocking for 1 hr in 2%
(v/v) BSA in PBS, grids were incubated with affinity-purified
LeSUT2 antibodies diluted 1:4 in PBS in a humid chamber at 48C
overnight. Sections were washed with PBS-T and PBS and were
incubated for 1 hr with 10-nm gold-coupled goat anti–rabbit IgG
(Amersham) diluted 1:50 in PBS. After three washes in PBS-T and
PBS, the sections were postfixed for 10 min in 2% glutaraldehyde in
PBS and rinsed with distilled wa- ter. Ultrathin sections were
poststained with uranyl acetate and lead citrate for better tissue
contrast. Gold labeling was detected with a Philips CM 100
microscope (Fellbach, Germany).
Mapping
Restriction fragment length polymorphism (RFLP) analysis and map-
ping of cDNA clones StSUT1, LeSUT4, and LeSUT2 were performed in
the diploid potato mapping population BC9162 of 67 individuals as
described (Gebhardt et al., 1991).
ACKNOWLEDGMENTS
We thank W.N. Fischer for pDura and SUSY7/ura3 and Lukas Bürkle for
help with LeSUT characterization. We are very grateful to
Nicole
Putative Sucrose Sensor 1163
Thiele and Katrin Herm for excellent technical assistance. This
work was supported by grants from Deutsche Forschungsgemeinschaft
(SFB446 B9).
Received February 10, 2000; accepted April 28, 2000.
REFERENCES
Alonso, J.M., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker,
J.R. (1999). EIN2, a bifunctional transducer of ethylene and stress
responses in Arabidopsis. Science 284, 2148–2152.
Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992). New
plant binary vectors with selectable markers located proximal to
the left T-DNA border. Plant Mol. Biol. 20, 1195–1197.
Boles, E., and Hollenberg, C.P. (1997). The molecular genetics of
hexose transport in yeasts. FEMS Microbiol. Rev. 21, 85–111.
Braun, V., and Killmann, H. (1999). Bacterial solutions to the
iron- supply problem. Trends Biochem. Sci., 24, 104–109.
Bürkle, L., Hibberd, J.M., Quick, W.P., Kühn, C., Hirner, B., and
Frommer, W.B. (1998). The H1-sucrose co-transporter NtSUT1 is
essential for sugar export from tobacco leaves. Plant Physiol. 118,
59–68.
Chiou, T.J., and Bush, D.R. (1998). Sucrose is a signal molecule in
assimilate partitioning. Proc. Natl. Acad. Sci. USA 95,
4784–4788.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified
method for Agrobacterium-mediated transformation of Arabidopsis
thal- iana. Plant J. 16, 735–743.
Gebhardt, C., Ritter, E., Barone, A., Debener, T., Walkemeier, B.,
Schachtschabel, U., Kaufmann, H., Thompson, R.D., Bonierbale, M.W.,
Ganal, M.W., Tanksley, S.D., and Salamini, F. (1991). RFLP maps of
potato and their alignment with the homeologous tomato genome.
Theor. Appl. Genet. 83, 49–57.
Gebhardt, C., Ritter, E., and Salamini, F. (1999). RFLP map of the
potato. In DNA-Based Markers in Plants, 2nd ed. R.L.V. Phillips,
I.K., ed (New York: Kluwer Academic Publishers).
Hellmann, H., Barker, L., Funck, D., and Frommer, W.B. (2000). The
regulation of assimilate allocation and transport. Aust. J. Plant
Physiol., in press.
Iraqui, I., Vissers, S., Bernard, F., De Craene, J.-O., Boles, E.,
Urrestarazu, A., and Andre, B. (1999). Amino acid signaling in
Saccharomyces cerevisiae: A permease-like sensor of external amino
acids and F-box protein Grr1p are required for transcrip- tional
induction of the AGP1 gene, which encodes a broad-speci- ficity
amino acid permease. Mol. Cell. Biol. 19, 989–1001.
Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS
fusions: b-Glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO J. 6, 3901–3907.
Kozak, M. (1996). Interpreting cDNA sequences: Some insights from
studies on translation. Mamm. Genome 7, 563–574.
Kühn, C., Quick, W.P., Schulz, A., Sonnewald, U., and Frommer, W.B.
(1996). Companion cell–specific inhibition of the potato sucrose
transporter SUT1. Plant Cell Environ. 19, 1115–1123.
Kühn, C., Franceschi, V.R., Schulz, A., Lemoine, R., and Frommer,
W.B. (1997). Localization and turnover of sucrose transporters
in
enucleate sieve elements indicate macromolecular trafficking.
Science 275, 1298–1300.
Kyte, J., and Doolittle, F.R. (1982). A simple method for
displaying the hydropathic character of a protein. J. Mol. Biol.
157, 693–698.
Lalonde, S., Boles, E., Hellmann, H., Barker, L., Patrick, J.W.,
Frommer, W.B., and Ward, J.M. (1999). A dual function of sugar
carriers: Transport and sugar sensing. Plant Cell 11,
707–726.
Larsson, C. (1985). Plasma Membranes. Vol. 1. H.F. Linskens and
J.F. Jackson, eds (Berlin: Springer Verlag).
Lemoine, R., Kühn, C., Thiele, N., Delrot, S., and Frommer, W.B.
(1996). Antisense inhibition of the sucrose transporter: Effects on
amount of carrier and sucrose transport activity. Plant Cell Envi-
ron. 19, 1124–1131.
Lemoine, R., Bürkle, L., Barker, L., Sakr, S., Kühn, C., Regnacq,
M., Gaillard, C., Delrot, S., and Frommer, W.B. (1999). Identifi-
cation of a pollen-specific sucrose transporter–like protein NtSUT3
from tobacco. FEBS Lett. 454, 325–330.
Liang, H., and Gaber, R.F. (1996). A novel signal transduction
path- way in Saccharomyces cerevisiae defined by SNF3-regulated
expression of HXT6. Mol. Biol. Cell 7, 1953–1966.
Martin, T., Hellmann, H., Schmidt, R., Willmitzer, L., and Frommer,
W.B. (1997). Identification of mutants in metabolically regulated
gene expression. Plant J. 11, 53–62.
Murashige, T., and Skoog, F. (1962). A revised medium for rapid
growth and bioassays with tobacco tissue culture. Physiol. Plant.
15, 473–497.
Özcan, S., and Johnston, M. (1995). Three different regulatory
mechanisms enable yeast hexose transporter (HXT) genes to be
induced by different levels of glucose. Mol. Cell. Biol. 15, 1564–
1572.
Özcan, S., Dover, J., Rosenwald, A.G., Woelfl, S., and Johnston, M.
(1996a). Two glucose transporters in S. cerevisiae are glucose
sensors that generate a signal for induction of gene expression.
Proc. Natl. Acad. Sci. USA 93, 12428–12432.
Özcan, S., Leong, T., and Johnston, M. (1996b). Rgt1p of Saccha-
romyces cerevisiae, a key regulator of glucose-induced genes, is
both an activator and a repressor of transcription. Mol. Cell.
Biol. 16, 6419–6426.
Özcan, S., Dover, J., and Johnston, M. (1998). Glucose sensing and
signaling by two glucose receptors in the yeast Saccharomy- ces
cerevisiae. EMBO J. 17, 2566–2573.
Rentsch, D., Laloi, M., Rouhara, I., Schmelzer, E., Delrot, S., and
Frommer, W. (1995). NTR1 encodes a high affinity oligopeptide
transporter in Arabidopsis. FEBS Lett. 370, 264–268.
Riesmeier, J.W., Willmitzer, L., and Frommer, W.B. (1992). Isola-
tion and characterization of a sucrose carrier cDNA from spinach by
functional expression in yeast. EMBO J. 11, 4705–4713.
Riesmeier, J., Hirner, B., and Frommer, W.B. (1993). Potato sucrose
transporter expression in minor veins indicates a role in phloem
loading. Plant Cell 5, 1591–1598.
Riesmeier, J.W., Willmitzer, L., and Frommer, W.B. (1994). Evi-
dence for an essential role of the sucrose transporter in phloem
loading and assimilate partitioning. EMBO J. 13, 1–7.
Roblin, G., Sakr, S., Bonmort, J., and Delrot, S. (1998).
Regulation of a plant plasma membrane sucrose transporter by
phosphoryla- tion. FEBS Lett. 424, 165–168.
1164 The Plant Cell
Roitsch, T. (1999). Source–sink regulation by sugar and stress.
Curr. Opin. Plant Biol. 2, 198–206.
Sauer, N., and Stolz, J. (1994). SUC1 and SUC2: Two sucrose
transporters from Arabidopsis thaliana; expression and character-
ization in baker’s yeast and identification of the histidine-tagged
protein. Plant J. 6, 67–77.
Schäfer-Pregl, R., Ritter, E., Concilio, L., Hesselbach, J.,
Lovatti, L., Walkemeier, B., Thelen, H., Salamini, F., and
Gebhardt, C. (1998). Analysis of quantitative trait loci (QTL) and
quantitative trait alleles (QTA) for potato tuber yield and starch
content. Theor. Appl. Genet. 97, 834–846.
Schwacke, R., Grallath, S., Breitkreuz, K.E., Stransky, E.,
Stransky, H., Frommer, W.B., and Rentsch, D. (1999). LeProT1, a
trans- porter for proline, glycine betaine, and g-butyric acid in
tomato pollen. Plant Cell 11, 377–392.
St. Johnston, D. (1995). The intracellular localization of
messenger RNAs. Cell 81, 161–170.
Stolz, J., Ludwig, A., Stadler, R., Biesgen, C., Hagemann, K., and
Sauer, N. (1999). Structural analysis of a plant sucrose carrier
using monoclonal antibodies and bacteriophage lambda surface
display. FEBS Lett. 453, 375–379.
Swofford, D.L. (1998). PAUP*. Phylogenetic Analysis Using Parsi-
mony (*and Other Methods). (Sunderland, MA: Sinauer
Associates).
Tadege, M., Bucher, M., Stähli, W., Suter, M., Dupuis, I.,
and
Kuhlemeier, C. (1998). Activation of plant defense responses and
sugar efflux by expression of pyruvate decarboxylase in potato
leaves. Plant J. 16, 661–671.
Vagnoli, P., Coons, D.M., and Bisson, L.F. (1998). The C-terminal
domain of Snf3p mediates glucose-responsive signal transduction in
Saccharomyces cerevisiae. FEMS Microbiol. Rev. 160, 31–36.
van Bel, A.J.E. (1996). Interaction between sieve element and com-
panion cell and the consequences for photoassimilate distri-
bution. Two structural hardware frames with associated
physiological software packages in dicotyledons. J. Exp. Bot. 47,
1129–1140.
Weise, A., Barker, L., Kühn, C., Lalonde, S., Buschmann, H.,
Frommer, W.B., and Ward, J.M. (2000). A new subfamily of sucrose
transporters SUT4 with low affinity/high capacity local- ized in
enucleate sieve elements of plants. Plant Cell (in press).
Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg,
C., and Boles, E. (1999). Concurrent knock-out of at least 21
transporter genes is required to block uptake of hexoses in yeast.
FEBS Lett. 464, 123–128.