Plant Physiol. (1998) 118: 59–68
The H1-Sucrose Cotransporter NtSUT1 Is Essential for Sugar
Export from Tobacco Leaves1
Lukas Bürkle, Julian M. Hibberd2, W. Paul Quick, Christina Kühn, Brigitte Hirner, and Wolf B. Frommer*
Botanical Institute, Eberhard Karls University, Auf der Morgenstelle 1, D-72076 Tübingen, Germany (L.B., C.K.,
B.H., W.B.F.); and Department of Animal and Plant Sciences, University of Sheffield,
Sheffield S10 2TN, United Kingdom (J.M.H., W.P.Q.)
In many species translocation of sucrose from the mesophyll to
the phloem is carrier mediated. A sucrose/H1-symporter cDNA,
NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown
to be highly expressed in mature leaves and at low levels in other
tissues, including floral organs. To study the in vivo function of
NtSUT1, tobacco plants were transformed with a SUT1 antisense
construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly
affected plants developed chloroses and necroses that led to death.
The leaves exhibited impaired ability to export recently fixed 14CO2
and were unable to export transient starch during extended periods
of darkness. As a consequence, soluble carbohydrates accumulated
and photosynthesis was reduced. Autoradiographs of leaves show a
heterogenous pattern of CO2 fixation even after a 24-h chase. The
14
C pattern does not change with time, suggesting that movement of
photosynthate between mesophyll cells may also be impaired. The
affected lines show a reduction in the development of the root
system and delayed or impaired flowering. Taken together, the
effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic
route and possibly for intermesophyll transport as well.
Photosynthesis in mature leaves produces a surplus of
assimilates. Carbohydrates derived from mature leaves are
distributed in the plant through the vascular system,
mainly in the form of sucrose, to support the growth of
heterotrophic tissues such as developing leaves, apices,
roots, and reproductive organs. Both active transport by
specific carriers across the plasma membrane and symplastic transport via plasmodesmata have been discussed as
possible mechanisms for phloem loading (Ap Rees, 1994).
Nevertheless, a direct demonstration of the actual role of
plasmodesmata in assimilate transport is still missing. Sucrose transport activities have been identified in a number
of plant species (for reviews, see Bush, 1993; Frommer et
al., 1996) and have been described as sucrose:proton co1
This work was supported by grants from the Biotechnology
and Biological Science Research Council (no. 50/PO1777) and the
Deutsche Forschungsgemeinschaft (SPP, Elevated CO2 and Transport, no. Fr989/5-2).
2
Present address: Department of Plant Sciences, University of
Cambridge, Downing Street, Cambridge, CB2 3EA UK.
* Corresponding author; e-mail [email protected]; fax
49 –7071–293287.
transport with a 1:1 stoichiometry (Bush, 1990; Lemoine et
al., 1996).
To resolve the question of whether carrier-mediated sucrose transport represents an essential step in phloem loading, the respective genes were identified. A yeast strain
was modified so that it could be used as a complementation system to isolate the SUT cDNAs SUT1 from spinach
and potato (Solanum tuberosum) (Riesmeier et al., 1992,
1993). Subsequently, homologous genes were isolated from
a number of other plant species (Gahrtz et al., 1994; Sauer
and Stolz, 1994; Weig and Komor, 1996; Hirose et al., 1997;
Kühn et al., 1997; Weber et al., 1997).
The biochemical properties of the transporters when expressed in yeast were similar to those described in protoplasts or in plasma membrane vesicles from a variety of
plant species. Detailed electrophysiological analyses in Xenopus oocytes demonstrated that SUT1 and SUC2 function
as sucrose:proton co-transporters (Boorer et al., 1996; Zhou
et al., 1997). The transporters are highly hydrophobic proteins and belong to a class of metabolite transporters consisting of two sets of six membrane-spanning regions separated by a central cytoplasmic loop (Ward et al., 1997).
SUT1 expression is highest in mature leaves and is subject to regulation by plant hormones (Harms et al., 1994).
Immunolocalization studies show that SUT1 is present at
high levels in the plasma membrane of sieve elements
(Kühn et al., 1997). Antisense repression of the SUT in
potato provided evidence for an essential role in phloem
loading (Riesmeier et al., 1994; Kühn et al., 1996). However,
potato is unusual in that it has been selected for high tuber
yields and is almost exclusively propagated asexually via
tubers. To investigate the role of SUT1 in a species that has
been selected for large leaf area and that propagates exclusively via seeds, a SUT1 cDNA was isolated from tobacco
(Nicotiana tabacum) and the expression pattern was characterized. Furthermore, transgenic tobacco plants were created with a reduction in the amount of SUT mRNA due to
antisense inhibition. These plants were analyzed with respect to the effects on export capacity, carbohydrate partitioning, photosynthesis, and plant development.
Abbreviations: CaMV, cauliflower mosaic virus; SUT, sucrose
transporter.
59
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60
Bürkle et al.
MATERIALS AND METHODS
Recombinant DNA
Recombinant phages (5 3 105) of a cDNA library derived
from tobacco (Nicotiana tabacum) leaves (Stratagene) were
screened with a 32P-labeled StSUT1 fragment (Riesmeier et
al., 1993). Positive clones were obtained and sequenced. For
reverse-transcriptase PCR, total RNA was extracted from
mature leaves (Riesmeier et al., 1993). Poly(A1) RNA was
purified via Dynabeads Oligo (dT)25 (Dynal Inc., Lake Success, NY). Poly(A1) RNA was reverse transcribed using
primer T1 (NCC-RAA-YAA-RTC-RTC-CAA-NGG-NCCNCC). The resulting first-strand cDNA was used as a
template for PCR amplification with T2 (GCN-GCN-GGNGTN-CAR-TTY-GGN-TGG-GCN) and T3 (GAA-AATATA-GAT-GCC-AAA-GCA-AAT-GG) for isolation of the
1.2-kb NtSUT1 fragment. The 59 end of NtSUT1 was isolated using the 59-AmpliFINDER RACE Kit (Clontech Laboratories, Palo Alto, CA). The amplified DNA fragments
were cloned into pON 184 (pACYC184 derivative with
Bluescript polylinker; O. Ninnemann and W.B. Frommer,
unpublished data) that had been previously cut with SmaI.
The 1.2-kb SmaI/SalI fragment of NtSUT1 was ligated in
reverse orientation between the CaMV 35S promoter and
the polyadenylation signal of the octopine-synthase gene
into the SmaI/SalI restriction site of the binary vector Bin 19
(Bevan, 1984).
Transformation and Analysis of Transgenic Plants
Transfer of the chimeric construct into Agrobacterium
tumefaciens GV2260 and transformation of tobacco cv SNN
were performed as described previously (Köster-Töpfer et
al., 1989). Transgenic plants used for molecular characterization were transferred to soil and analyzed under greenhouse conditions. For northern-blot analysis, RNA was
isolated from mature leaves of greenhouse-grown transformants and wild-type plants after 4 to 6 h of light, as
described previously (Riesmeier et al., 1993). Northern
blots were made under stringent conditions, hybridized in
50% formamide at 42°C, and washed with 23 SSC at 68°C.
Transgenic lines were named aNtSUT1-35S. About onehalf of the transformants derive from a comparable construct, the only difference being that a triple CaMV 35S
enhancer was used (marked with an asterisk). Seeds were
collected from selected lines (wild type, aNtSUT1-35S17,
aNtSUT1-35S30, and aNtSUT1-35S55*) for further analysis.
Offspring were proven to contain the SUT1-antisense construct by a PCR strategy previously described by Hamill et
al. (1991) (data not shown).
Physiological Measurements
Plants used for growth analysis, export-rate measurements, photosynthesis, and assessment of the concentration of soluble carbohydrate and starch were grown for 10
to 12 weeks in controlled-environment cabinets. Light intensity was 500 mmol m22 s21 with a 14-h photoperiod and
a 25°C/20°C day/night temperature cycle. Seeds were germinated on sand in Petri dishes for 12 d before transplan-
Plant Physiol. Vol. 118, 1998
tation. Plants were grown in sand and supplied with Rorison’s nutrient solution containing 2.2 mm nitrogen (Hewitt,
1966). Photosynthetic rates of the youngest fully expanded
leaf were measured by IR gas analysis using a portable
analyzer (model LCA3, Analytical Development Co., Ltd.,
Hoddeston, UK) at growth irradiance.
For analysis of carbohydrates, leaf discs were taken from
mature leaves 5 h into the photoperiod and placed in liquid
nitrogen, and soluble sugars were extracted in 80% buffered ethanol (5 mm MgCl2, 50 mm Hepes, pH 7.4) at 70°C.
Starch was extracted in sodium acetate buffer, pH 4.7,
containing a-amylase and amyloglucosidase according to
the method of Stitt et al. (1989). Soluble carbohydrates were
assayed enzymatically as described by Stitt et al. (1989).
Feeding of 14CO2 and partitioning of 14C were performed
according to the method of Quick et al. (1989). Plants used
for growth analysis were separated into shoot and root and
then dried in a forced-air oven at 80°C until a constant dry
weight was attained.
To assess the ability of leaves to export carbon, leaves
were enclosed in a cuvette containing a Geiger-Müller tube
positioned directly under the leaf. The light intensity at the
cuvette surface was maintained at the same level as that
used for plant growth and the temperature was kept at
25°C. Leaves were left to stabilize for 2 h; at 11:00 am each
day, 14CO2 was supplied to each leaf for 5 min according to
the method of Hibberd et al. (1996). The rate at which 14C
disappeared from each leaf was then recorded on a chart
recorder during the photoperiod. The data were fitted to a
double exponential curve qt(t) 5 A1e2l1t 1 A2e2l2t 1 B,
where qt is the total amount of isotope incorporated, t is
time, A1 and A2 are the values at 0 time of the two exponentials, l1 and l2 are the coefficients of the two exponentials, and B is the value of the asymptote set from the
measured proportion of 14C incorporated into starch (see
above).
The transfer coefficient for phloem loading was then
calculated from k01 5 (A1l1 1 A2l2)/(A1 1 A2) according to
the method of Rocher and Prioul (1987). While 14C was fed
to the leaf and its export was monitored, the rate of net
photosynthesis of the leaf was measured by linking the
cuvette to an IR gas analyzer (Analytical Development Co.,
Ltd.). The whole plant was illuminated during the light
period with tungsten/halogen lamps identical to those
provided in the growth cabinet. Leaves comparable to
those used for export were sampled to measure the concentration of sucrose at 11:00 am. Export rates from the
leaves were calculated from the measured concentration of
sucrose and the transfer coefficient calculated from the 14C
export curves. For autoradiography, leaves were supplied
with 14CO2 in the same way as for export experiments, but
a larger leaf cuvette was used (20 3 30 cm). Leaves were
detached from the plant either immediately or 24 h after
feeding, and immediately frozen in dry ice followed by
exposure to radiographic film for 7 d.
RESULTS
An antisense approach was used to study the physiological role of SUT1 in tobacco. Since tobacco and potato
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Suc Transport in Tobacco
61
cDNA and subsequent PCR with the anchor primer and a
specific primer derived from the central fragment.
Consistent with the amphidiploidy of tobacco, two subclasses of 59 clones with slight differences in the untranslated regions were identified. A hypothetical sequence
composed of one of the 59 sequences, the central fragment,
and the overlapping 39 sequence was constructed and deposited with the second 59 end in the EMBL database
(accession nos. X82276 and X82277). A computer-aided
analysis of homologies between SUTs from different species shows that NtSUT1 is most similar to the potato
StSUT1, indicating that it may serve a similar function
(phloem loading) in tobacco as StSUT1 serves in potato
(Fig. 1).
Expression of SUT1 in Tobacco
Figure 1. Computer-aided homology analysis by PHYLIP (Felsenstein, 1993) of aligned SUTs from tobacco (NtSut1), potato (StSut1)
(Riesmeier et al., 1993), spinach (SoSut1) (Riesmeier et al., 1992),
Arabidopsis (AtSuc1 and AtSuc2) (Sauer and Stolz, 1994), Plantago
major (PmSuc1 and PmSuc2) (Gahrtz et al., 1994, 1996), castor bean
(RcSut1) (Weig and Komor, 1996), rice (OsSut1) (Hirose et al., 1997),
and fava bean (VfSut1) (Weber et al., 1997). The comparison was
restricted to the region in NtSUT1 from amino acid position 19 to
472. The numbers indicate the occurrence of a branch in 100
bootstrap replicates of a given data set. OsSut1 was used as the
outgroup.
(Solanum tuberosum) are closely related species, and therefore respective genes were likely to be highly homologous,
tobacco plants were transformed with the antisense construct used to inhibit phloem loading in potato (Riesmeier
et al., 1994). However, none of the 60 screened transformants showed reduced RNA levels or characteristic phenotypic symptoms (W.B. Frommer, unpublished results).
To determine whether this was attributable to the different
physiology of tobacco plants or to technical problems associated with the lack of sufficient homology between the
potato and tobacco genes, the orthologous SUT1 gene was
isolated from tobacco.
The central 1.2-kb fragment of NtSUT1 was used to
analyze SUT1 mRNA levels in different tobacco organs.
NtSUT1 was found to be highly expressed in mature
leaves, whereas expression was low in developing sink
leaves (Fig. 2A). Expression was also found in all other
tissues analyzed, i.e. roots, stems, and all flower organs
(Fig. 2, A and B). The ubiquitous expression may indicate
that the transporter is important not only for phloem loading, but also for retrieval along the translocation path
and/or for unloading processes. These results are consistent with immunolocalization and GUS expression data of
SUT1 in tobacco (Kühn et al., 1997; B. Hirner and W.B.
Frommer, unpublished results). Nevertheless, we cannot
fully exclude the possibility of cross-hybridization with
other, as-yet-unknown SUT mRNAs in tobacco.
Antisense Repression of the SUT
To determine the physiological role of SUT1, NtSUT1
was cloned in an antisense orientation behind the CaMV
35S promoter into a binary vector (Fig. 3). Leaf discs of
tobacco were transformed with the chimeric construct and,
after transfer of the regenerated plants to the greenhouse,
71 of 91 transformants showed no visible phenotypic pe-
Isolation of Tobacco SUT Genes
A tobacco leaf cDNA library was screened using the
potato SUT1 cDNA as a probe. Eight clones contained
inserts of about 450 bp encoding the 39 terminal sequence
of NtSUT1. To obtain larger fragments via reversetranscriptase PCR, conserved sequences were identified by
sequence comparisons of spinach, potato, and Arabidopsis
SUT cDNAs, and were then used to generate degenerate
oligonucleotides (Riesmeier et al., 1992, 1993; Sauer and
Stolz, 1994). First-strand cDNA from mature leaves primed
with the degenerate primer T1 was used as a template for
two degenerate primers (T2 and T3) to amplify a central,
1245-bp NtSUT1 fragment. The missing 59 end was obtained after ligation of an anchor onto the first-strand
Figure 2. Northern-blot analysis of NtSUT1 expression in tobacco
using stringent conditions (see “Materials and Methods”; probe,
NtSut1, 1.2 kb). A, Expression in various parts of the plant (16.5
mg/lane). B, Expression in flower organs (5 mg/lane).
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62
Bürkle et al.
Plant Physiol. Vol. 118, 1998
was lower in lines aNtSUT1-35S30 and aNtSUT1-35S55*
compared with the wild type (Fig. 5C).
Biochemical and Physiological Analysis of SUT
Antisense Plants
Retarded development and the phenotype of plants containing reduced amounts of SUT1 mRNA indicated that
Figure 3. Structure of the chimeric gene and analysis of transgenic
tobacco plants with reduced expression of NtSUT1. Analysis of
NtSUT1 mRNA expression in source leaves of transgenic and control
plants. Total RNA (25 mg/lane) was hybridized with a radiolabeled
NtSUT1 1.2-kb probe under stringent conditions (see “Materials and
Methods”). Transcript sizes are given on the right. The hybridization
signals at 1500 nt represent the antisense NtSUT1 mRNA.
culiarities compared with the wild type. However, 10
plants developed a weak phenotype (e.g. aNtSUT1-35S46*),
whereas 10 other plants clearly showed visible symptoms,
as described below. aNtSUT1-35S12 and aNtSUT1-35S50
were the most strongly affected plants, whereas aNtSUT135S17, aNtSUT1-35S19, aNtSUT1-35S20, aNtSUT1-35S29,
aNtSUT1-35S30, aNtSUT1-35S33, aNtSUT1-35S34, and
aNtSUT1-35S55* were only intermediately affected. Several
of these plants were tested at the RNA level using the
central, 1.2-kb fragment of NtSUT1 as a probe. Only plants
displaying symptoms (aNtSUT1-35S12, aNtSUT1-35S17,
aNtSUT1-35S19, aNtSUT1-35S20, and aNtSUT1-35S33)
showed a significant reduction in NtSUT1 mRNA (Fig. 3),
whereas unaffected plants such as aNtSUT1-35S7,
aNtSUT1-35S26, and aNtSUT1-35S43 had SUT1 mRNA levels similar to those of the wild type.
Phenotypic Effects of SUT Inhibition
aNtSUT1-35S12 and aNtSUT1-35S50 were the most
strongly affected plants. During maturation, leaves began
to curl and rims and intercostal fields became chlorotic or
even necrotic (Fig. 4, A and B). In contrast, sink leaves
appeared unaffected. Both lines exhibited retarded leaf
development and grew no taller than 10 cm. At this stage,
development was arrested, without flower induction for
longer than 12 months. aNtSUT1-35S55* was slightly less
affected, also retarded in growth but eventually flowering
and setting seeds (data not shown).
Three seed-producing transformants with weak to intermediate phenotypes, aNtSUT1-35S17, aNtSUT1-35S30, and
aNtSUT1-35S55*, were grown in a controlled-growth
environment and used for more detailed physiological
analyses. In the controlled environment, these plants
also showed the above-described phenotype (Fig. 5A).
Dry-weight accumulation in lines aNtSUT1-35S30 and
aNtSUT1-35S55* was greatly reduced, with line aNtSUT135S17 being intermediate in its growth response relative to
lines aNtSUT1-35S30, aNtSUT1-35S55*, and the wild type
(Fig. 5B). The same gradation was also seen in the shootto-root ratio; the amount of root produced per unit of shoot
Figure 4. Development of symptoms in tobacco plants transformed
with the SUT1 antisense construct. A, Transgenic tobacco plants after
7 weeks in the greenhouse (from left to right): wild type, aNtSUT135S33, aNtSUT1-35S30, and aNtSUT1-35S12. B, Top view of wild
type (left) and aNtSUT1-35S50 (right). C, Starch accumulation as
determined after 16 h of darkness for wild type (control), aNtSUT135S30, and aNtSUT1-35S12 by KI staining.
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Suc Transport in Tobacco
63
Figure 5. Growth response of three selected antisense plants with weak to intermediate phenotypes. A, Phenotype; B, dry
weight; C, shoot-to-root ratio; and D, photosynthesis of tobacco plants transformed with the SUT1 antisense construct. Rate
of net photosynthesis of the youngest fully expanded leaf was measured at a growth irradiance of 500 mmol photons m22
s21. wt, Wild type; 17, aNtSUT1-35S17; 30, aNtSUT1-35S30; 55*, aNtSUT1-35S55*.
photosynthesis was affected and the export of carbohydrates from leaves was impaired. The rate of net photosynthesis at growth irradiance was reduced in all lines relative
to controls, with the reduction in photosynthesis being
greatest in lines showing the largest reduction in growth
and the strongest phenotype (Fig. 5D; Table I). To deterTable I. Rate of sucrose export from mature leaves of wild type
(WT), aNtSUT1-35S17 (17), aNtSUT1-35S30 (30), and aNtSUT135S55* (55)
Export was calculated from the measured rate of net photosynthesis, the measured sucrose concentration in the leaf, and the transfer
coefficient (K01). For details, see “Materials and Methods.”
Plant
WT
17
30
55*
Rate of Net
Photosynthesis
Sucrose
K01
Export Rate
mmol m22 s21
mmol m22
m s 21
mmol m22
s 21
14.1
11.8
6.7
4.3
3.8
4.0
12.5
21.5
975
844
50
6
3.7
3.3
0.6
0.1
mine directly whether the lower rates of sucrose transport
from the leaves was responsible for reduced growth, rates
of export of assimilate from leaf tissue, primarily sucrose,
were analyzed (Fig. 6). 14CO2 was fed to leaves and the
export of the fixed 14C was subsequently monitored. In
wild-type plants the initial 14C export occurred at a fast
rate, subsequently decreasing progressively. Export of 14C
from wild-type leaves fitted well to a double-exponential
equation (see “Materials and Methods”).
Both developing and mature leaves of aNtSUT1-35S17,
aNtSUT1-35S30, and aNtSUT1-35S55* exported less radiolabeled carbon than the wild type (Fig. 6). Inhibition of 14C
export became more pronounced as leaves matured (Fig.
6). Both the initial slope of 14C export and the second,
slower phase of 14C export were lower in aNtSUT1-35S17,
aNtSUT1-35S30, and aNtSUT1-35S55* than in the wild type.
The rate of sucrose export was calculated from the rate of
photosynthesis of the leaves used for efflux, from the sucrose concentrations measured in the leaves, and from the
transfer coefficients calculated from the export data of
mature leaves. Despite the fact that the concentration of
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64
Bürkle et al.
Plant Physiol. Vol. 118, 1998
of chlorophyll distribution shown in older chlorotic leaves
(Fig. 7). There was no evidence of chlorosis, however, in the
younger leaves chosen for this experiment (Fig. 8D). There
was also no evidence of export of label after a 24-h chase or
of reallocation of label within the leaf, with 14C remaining
localized around the major veins (Fig. 8F).
Rates of 14CO2 incorporation into leaf discs were comparable to the rates of photosynthesis measured by IR gas
analysis; both were reduced in antisense plants compared
with wild-type controls (Table II). Partitioning of recently
fixed photosynthate between the insoluble starch pool and
the soluble sucrose pool, however, was unaffected in the
transgenic lines. Thus, the observed reduction in photosynthesis was associated with a parallel reduction of both
starch and sucrose synthesis (Table II).
The concentration of soluble sugars increased in all three
antisense lines and was greatest in line aNtSUT1-35S55*
(Fig. 9, A–C). The concentration of sucrose and starch
increased in the regions proximal and distal to major veins
as carbon export was inhibited (Fig. 9, A and D). In lines
aNtSUT1-35S30 and a NtSUT1-35S55*, Glc and Fru clearly
accumulated in regions distal to major veins (Fig. 9, B
and C).
Figure 6. The export of 14C from leaves of tobacco. Wild-type plants
(F) and the transgenic lines aNtSUT1-35S17 (‚), aNtSUT1-35S30 (E),
and aNtSUT1-35S55* (M) expressing antisense NtSUT1 mRNA. A,
Data from young leaves; B, data from mature, fully expanded leaves.
Leaves were fed with 14CO2 at 11:00 AM and the export of 14C was
followed with a Geiger-Müller tube positioned under the fed area of
the leaf. Data are expressed as the maximum amount of isotope
incorporated. The black bar on the x axis represents the dark period.
sucrose increased in the leaves of antisense plants, the rate
of sucrose export from those leaves was reduced compared
with the wild type (Table I).
After the leaves of lines expressing antisense mRNA
reached full expansion, a progressive development of chlorosis was observed in interveinal regions. The yellow sectoring started at the tips and moved to the base as the
leaves aged, following a sink-to-source transition pattern
(Fig. 7). All parts of wild-type leaves fixed 14CO2 relatively
homogenously and then exported the majority of the isotope supplied after a 24-h chase period (Fig. 8, A–C). However, for transgenic lines (data shown for aNtSUT1-35S55*),
the distribution of 14CO2 fixation was heterogenous after 5
min of photosynthesis (Fig. 8E) and resembled the pattern
Figure 7. Typical pattern of chlorosis observed in the interveinal
regions of mature leaves from line aNtSUT1-35S30. This phenotype
developed progressively after leaves attained full expansion and only
in lines exhibiting a marked inhibition of sucrose export.
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Suc Transport in Tobacco
Figure 8. Photographs (A and D) and autoradiographs (B, C, E, and
F) of wild-type (A–C) and transgenic (D–F) plants. Leaves were allowed to fix 14CO2 for 5 min and were then detached from the plant
(B and E) or allowed to remain attached for another 24 h to permit
export of incorporated radioactivity (C and F).
After they were kept in darkness for 16 h, wild-type
plants had only low levels of starch, whereas antisense
plants showed strong iodine staining, indicating that they
were unable to mobilize excess carbohydrates accumulated
during the previous day (Fig. 4C).
DISCUSSION
In general, two potential routes have been discussed for
the loading of phloem with assimilates: the symplastic and
apoplastic pathways. In terms of the classification of plants
into symplastic or apoplastic loaders, Solanaceae represents a polytypic family encompassing species with relatively high plasmodesmata density between mesophyll and
the sieve-element/companion-cell complex (intermediate
type 1–2a; Datura, 1–10 plasmodesmata mm22 interface)
and species with low plasmodesmal connectivity (closed
primitive type 2a; tobacco, 0.12 plasmodesmata mm22 in-
65
terface; potato, 0.08 plasmodesmata mm22 interface; Gamalei, 1991). These data are supported by a detailed analysis
of individual vein structure from potato leaves (McCauley
and Evert, 1989).
A protein that could be responsible for phloem loading
with sucrose was identified from potato via yeast complementation (Riesmeier et al., 1993). The tissue specificity
(prevalence in the phloem), together with an observed
increase in sucrose-transport activity during the sink-tosource transition of sugar beet leaves and the increase of
SUT1 mRNA during leaf development from sink to source
in potato, were taken as strong indications that the SUT is
involved in phloem loading (Lemoine et al., 1992; Riesmeier et al., 1993).
Direct evidence for an apoplastic route of phloem loading in potato came from analysis of potato plants in which
sucrose transport was inhibited by antisense repression of
SUT1 (Riesmeier et al., 1994; Kühn et al., 1996; Lemoine et
al., 1996). Inhibited plants were retarded in development
and showed severe symptoms in mature leaves indicative
of osmotic problems associated with carbohydrate accumulation. Furthermore, sucrose export from leaves was inhibited, leading to reduced root development and dramatically reduced tuber yield.
To study the role of SUT1 in tobacco, transgenic tobacco
plants containing reduced amounts of endogenous NtSUT1
mRNA due to antisense inhibition were generated. Twenty
of ninety-one transformants had reduced amounts of SUT1
mRNA and also displayed characteristic symptoms of leaf
curling and chlorosis.
Feeding 14CO2 allowed us to monitor 14C export from
leaves of aNtSUT1-35S17, aNtSUT1-35S30, aNtSUT1-35S55*,
and wild-type plants. In wild-type plants the export of 14C
from leaves occurred in two clear phases, fitting well to a
double-exponential equation (see “Materials and Methods”). The export characteristics of lines aNtSUT1-35S30
and aNtSUT1-35S55* were markedly different from those of
the wild type, whereas aNtSUT1-35S17 showed an intermediate response; all lines showed decreased export compared with the wild type. The degree to which 14C export
was inhibited in the antisense lines increased as the leaves
matured.
Export of radiolabeled carbon from leaves has previously
been shown to occur in at least two phases and to fit well
to exponential functions (Rocher and Prioul, 1987). Compartmental analysis of export of photosynthetically fixed
14
C from mature leaves should allow pool sizes and rates of
Table II. Rate of incorporation of 14CO2 and the partitioning of 14C into water-soluble and -insoluble fractions of leaf discs from wild-type
(WT), aNtSUT1-35S17 (17), aNtSUT1-35S30 (30), and aNtSUT1-35S55* (55)
14
CO2 was fed at growth light intensity.
Plant
WT
17
30
55*
Rate of
Total
15.3 6 0.8
12.8 6 0.6
12.0 6 1.1
11.3 6 1.2
14
C Incorporation
Soluble fraction
mmol m22s21
7.3 6 0.2
6.4 6 0.3
5.7 6 0.4
5.6 6 0.6
Insoluble fraction
7.9 6 0.7
6.4 6 0.3
5.6 6 0.8
4.6 6 0.6
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Ratio
1.07 6 0.08
1.01 6 0.03
0.99 6 0.03
0.96 6 0.11
66
Bürkle et al.
Plant Physiol. Vol. 118, 1998
Figure 9. Concentration of soluble sugars and
starch in leaves from tobacco plants transformed
with antisense constructs of the SUT. Samples
were taken 5 h into the photoperiod. WT, Wild
type; 17a, aNtSUT1-35S17; 30, aNtSUT1-35S30;
55, aNtSUT1-35S55*. Data are means 6 SE.
Open bars, Regions distal to major veins;
shaded bars, equal regions proximal to major
veins.
export of carbon from leaves to be calculated, but compartmental analysis relies on a set of assumptions (Rocher and
Prioul, 1987; Zierler, 1981). Most of the assumptions do not
hold in the antisense lines, and therefore pool sizes were
not estimated from our export data. By measuring the
concentration of sucrose in the leaves biochemically, the
rate of sucrose export could be estimated (Table I). However, these data are likely to overestimate the rates of
export, because sucrose is typically present in more than
one pool in a leaf and only one pool will be directly
available for export (Rocher and Prioul, 1987). Despite the
fact that sucrose export from these leaves may have been
overestimated because of its accumulation, the rates of
export were still much lower than in the wild type, strongly
supporting the proposed role of NtSUT1 in sucrose export
from tobacco leaves.
The proportion of 14CO2 partitioned between sucrose
and starch could also affect the rate and amount of 14C
exported from a leaf. By measuring the short-term partitioning of 14C between soluble and insoluble fractions, it
was shown that slower rates of carbon export from
aNtSUT1-35S17, aNtSUT1-35S30, and aNtSUT1-35S55* were
not due to a stimulation of the production of starch (Table
II). Despite the fact that mature leaves of lines aNtSUT135S30 and aNtSUT1-35S55* exported much less than leaves
of the wild type, the reduction in carbohydrate export was
not lethal. However, plants with more pronounced phenotypes, such as aNtSUT1-35S12, exhibited stronger inhibition of phloem loading and did not produce seed.
Sugars accumulated in plants in which expression of
antisense NtSUT1 mRNA led to reduced export of carbon
from leaves. Accumulation of soluble sugars and starch in
response to inhibition of carbon export from leaves is well
documented (von Schaewen et al., 1990; Krapp et al., 1991;
Kühn et al., 1996). However, in lines aNtSUT1-35S30 and
aNtSUT1-35S55*, there was a clear difference between the
concentration of hexoses distal and proximal to major
veins.
Typically, when carbohydrates accumulate in leaves,
down-regulation of photosynthesis occurs (Jang and Sheen,
1994). The rates of photosynthesis in aNtSUT1-35S17,
aNtSUT1-35S30, and aNtSUT1-35S55* were lower than in
the wild type. Furthermore, autoradiography showed that
even before development of the mottled, chlorotic phenotype, photosynthesis was concentrated in the regions close
to major veins and down-regulated in areas between the
veins. Areas where photosynthesis had become downregulated coincided with the areas where hexoses accumulated to high levels. Accumulation of hexoses has previously
been implicated in the down-regulation of photosynthetic
gene expression (Herbers et al., 1996).
These results further support the assumption that SUT1
is essential for sucrose export from leaves both during the
day and at night, since even when sugar biosynthesis was
reduced, soluble sugars accumulated and remained high
after extended periods of darkness, suggesting that the antisense plants were unable to export stored sugar reserves.
Similar results, including a mottled leaf phenotype, were
observed in detached spinach leaves fed carbohydrates
(Krapp et al., 1991), indicating that accumulation of carbohydrate alone could induce the change in leaf phenotype.
Molecular mechanisms that could bring about these changes
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Suc Transport in Tobacco
have been demonstrated and include carbohydrate-induced
repression of photosynthetic gene expression (Jang and
Sheen, 1994). High leaf starch is also associated with this
phenotype, but may not be a prerequisite for the appearance
of the symptoms in wild-type plants. In a previous study,
potato plants containing a similar antisense SUT1 construct
also had a similar mottled leaf phenotype; however, areas of
high or low photosynthesis did not correlate well with the
pattern of starch deposition (Kühn et al., 1996), and plant
growth was severely reduced, especially in the roots, resulting in a large increase in the shoot-to-root ratio. This agrees
with the hypothesis that carbohydrate export to heterotrophic tissues was impaired, suggesting an important role of
the SUT in tobacco for the export of sucrose from source
leaves.
It will be important to determine the remaining sucrose
transport activity in antisense plants to determine whether
SUT1 activity may become limiting for photosynthesis under optimal conditions, e.g. at increased atmospheric CO2
levels, thus leading to the typical acclimation phenomena
observed in various plants (Besford, 1990).
In summary, the results obtained with transgenic plants
in which the SUT was inhibited clearly show that SUT1 is
important for sucrose export from leaves, a strong antisense reduction being lethal. Because it is localized on
sieve-element plasma membranes in tobacco and potato
(Kühn et al., 1997), it is responsible for phloem loading
both in tobacco, which has a large leaf area relative to the
dry weight of reproductive organs, and in potato, which
has a high tuber-harvest index, and thus appears to be a
more general mechanism of phloem loading, at least for
type 2a plants. However, low levels of NtSUT1 expression
were also found in a number of different sink tissues,
including stems and parts of the flower, indicating that
SUT1 may have other functions, such as sucrose retrieval
along the translocation path and possibly phloem unloading. It will be important in the future to determine the
precise function of SUT1 expression in these tissues by
cell-type-specific antisense experiments. Because sucrose,
the major osmotic compound present in phloem sap, is
thought to create the major driving force for mass flow in
the phloem, antisense plants with reduced loading capacity
should provide an excellent tool with which to study the
mechanisms driving phloem translocation (Münch, 1930).
ACKNOWLEDGMENT
We are very grateful to Nicole Thiele for her excellent technical
assistance.
Received March 30, 1998; accepted June 19, 1998.
Copyright Clearance Center: 0032–0889/98/118/0059/10.
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