Plant Physiol. (1998) 118: 59-68
The H+-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.)
 |
ABSTRACT |
In many
species translocation of sucrose from the mesophyll to the phloem is
carrier mediated. A sucrose/H+-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 CO2 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 14C
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.
| |
INTRODUCTION |
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 cotransport 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.
| |
MATERIALS AND METHODS |
Recombinant DNA
Recombinant phages (5 × 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(A+) RNA was purified via Dynabeads Oligo
(dT)25 (Dynal Inc., Lake Success, NY).
Poly(A+) RNA was reverse transcribed using primer
T1 (NCC-RAA-YAA-RTC-RTC-CAA-NGG-NCC-NCC). The resulting first-strand
cDNA was used as a template for PCR amplification with T2
(GCN-GCN-GGN-GTN-CAR-TTY-GGN-TGG-GCN) and T3
(GAA-AAT-ATA-GAT-GCC-AAA-GCA-AAT-GG) for isolation of the 1.2-kb NtSUT1 fragment. The 5
end of NtSUT1 was
isolated using the 5-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 2× SSC at 68°C. Transgenic
lines were named 
NtSUT1-35S. About one-half 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,
NtSUT1-35S17, NtSUT1-35S30, and
NtSUT1-35S55*) 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 µmol
m
2 s1 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
transplantation. 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 -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) = A1e
1t + A2e2t + 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, 
1 and 2 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 = (A11 + A22)/(A1 + 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 CO2 in the same way as
for export experiments, but a larger leaf cuvette was used (20 × 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 (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.
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 3 terminal sequence of NtSUT1. To
obtain larger fragments via reverse-transcriptase 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 5 end was obtained after ligation of an anchor onto the
first-strand 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 5
clones with slight differences in the untranslated regions were
identified. A hypothetical sequence composed of one of the 5
sequences, the central fragment, and the overlapping 3 sequence was
constructed and deposited with the second 5 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).
View larger version (12K):
[in this window]
[in a new window]
| 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.
|
|
Expression of SUT1 in 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.
View larger version (35K):
[in this window]
[in a new window]
| 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 µg/lane). B, Expression in flower
organs (5 µg/lane).
|
|
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 peculiarities compared with
the wild type. However, 10 plants developed a weak phenotype (e.g.
NtSUT1-35S46*), whereas 10 other plants
clearly showed visible symptoms, as described below.
NtSUT1-35S12 and
NtSUT1-35S50 were the most strongly affected
plants, whereas NtSUT1-35S17,
NtSUT1-35S19,
NtSUT1-35S20, NtSUT1-35S29,
NtSUT1-35S30,
NtSUT1-35S33,
NtSUT1-35S34, and NtSUT1-35S55* 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 (NtSUT1-35S12,
NtSUT1-35S17,
NtSUT1-35S19,
NtSUT1-35S20, and NtSUT1-35S33) showed a significant reduction
in NtSUT1 mRNA (Fig. 3), whereas unaffected plants such as
NtSUT1-35S7,
NtSUT1-35S26, and
NtSUT1-35S43 had SUT1 mRNA levels
similar to those of the wild type.
View larger version (43K):
[in this window]
[in a new window]
| 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 µg/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.
|
|
Phenotypic Effects of SUT Inhibition
NtSUT1-35S12 and
NtSUT1-35S50 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.
NtSUT1-35S55* was slightly less affected, also
retarded in growth but eventually flowering and setting seeds (data not
shown).
View larger version (65K):
[in this window]
[in a new window]
| 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, NtSUT1-35S33,
NtSUT1-35S30, and NtSUT1-35S12. B, Top
view of wild type (left) and NtSUT1-35S50 (right). C,
Starch accumulation as determined after 16 h of darkness for wild
type (control), NtSUT1-35S30, and
NtSUT1-35S12 by KI staining.
|
|
Three seed-producing transformants with weak to intermediate
phenotypes, NtSUT1-35S17,
NtSUT1-35S30, and
NtSUT1-35S55*, 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 NtSUT1-35S30 and
NtSUT1-35S55* was greatly reduced, with line
NtSUT1-35S17 being intermediate in its growth
response relative to lines NtSUT1-35S30,
NtSUT1-35S55*, and the wild type (Fig. 5B).
The same gradation was also seen in the shoot-to-root ratio; the amount
of root produced per unit of shoot was lower in lines
NtSUT1-35S30 and
NtSUT1-35S55* compared with the wild type
(Fig. 5C).
View larger version (82K):
[in this window]
[in a new window]
| 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 µmol photons m2
s1. wt, Wild type; 17, NtSUT1-35S17; 30, NtSUT1-35S30; 55*, NtSUT1-35S55*.
|
|
Biochemical and Physiological Analysis of SUT Antisense Plants
Retarded development and the phenotype of plants containing
reduced amounts of SUT1 mRNA indicated that 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 determine 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 C
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'').
View this table:
[in this window]
[in a new window]
|
Table I.
Rate of sucrose export from mature leaves of wild
type (WT), NtSUT1-35S17 (17),
NtSUT1-35S30 (30), and NtSUT1-35S55* (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''.
|
|
View larger version (26K):
[in this window]
[in a new window]
| Figure 6.
The export of 14C from leaves of
tobacco. Wild-type plants ( ) and the transgenic lines
NtSUT1-35S17 ( ), NtSUT1-35S30 ( ),
and NtSUT1-35S55* ( ) 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.
|
|
Both developing and mature leaves of
NtSUT1-35S17,
NtSUT1-35S30, and
NtSUT1-35S55* 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
NtSUT1-35S17,
NtSUT1-35S30, and
NtSUT1-35S55* 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
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 CO2
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 NtSUT1-35S55*), the
distribution of 14CO2
fixation was heterogenous after 5 min of photosynthesis (Fig. 8E) and
resembled the pattern 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).
View larger version (103K):
[in this window]
[in a new window]
| Figure 7.
Typical pattern of chlorosis observed in the
interveinal regions of mature leaves from line
NtSUT1-35S30. This phenotype developed progressively
after leaves attained full expansion and only in lines exhibiting a
marked inhibition of sucrose export.
|
|
View larger version (162K):
[in this window]
[in a new window]
| 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).
|
|
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).
View this table:
[in this window]
[in a new window]
|
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), NtSUT1-35S17 (17),
NtSUT1-35S30 (30), and NtSUT1-35S55* (55)
14CO2 was fed at growth light intensity.
|
|
The concentration of soluble sugars increased in all three antisense
lines and was greatest in line NtSUT1-35S55*
(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
NtSUT1-35S30 and NtSUT1-35S55*, Glc and Fru clearly accumulated in
regions distal to major veins (Fig. 9, B and C).
View larger version (39K):
[in this window]
[in a new window]
| 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, NtSUT1-35S17; 30, NtSUT1-35S30; 55, NtSUT1-35S55*. Data are means ± SE.
Open bars, Regions distal to major veins; shaded bars, equal regions
proximal to major veins.
|
|
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
µm2 interface) and species with low
plasmodesmal connectivity (closed primitive type 2a; tobacco, 0.12 plasmodesmata µm2 interface; potato, 0.08 plasmodesmata µm2 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-to-source 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
NtSUT1-35S17,
NtSUT1-35S30,
NtSUT1-35S55*, 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 NtSUT1-35S30 and
NtSUT1-35S55* were markedly different from
those of the wild type, whereas NtSUT1-35S17
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 14C from mature leaves
should allow pool sizes and rates of 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 NtSUT1-35S17,
NtSUT1-35S30, and NtSUT1-35S55* were not due to a stimulation of
the production of starch (Table II). Despite the fact that mature
leaves of lines NtSUT1-35S30 and
NtSUT1-35S55* 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
NtSUT1-35S12, 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
NtSUT1-35S30 and
NtSUT1-35S55*, 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 NtSUT1-35S17,
NtSUT1-35S30, and
NtSUT1-35S55* 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 down-regulated
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 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).
| |
FOOTNOTES |
1
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 frommer{at}uni-tuebingen.de; fax
49-7071-293287.
Received March 30, 1998;
accepted June 19, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
SUT, sucrose transporter.
| |
ACKNOWLEDGMENT |
We are very grateful to Nicole Thiele for her excellent
technical assistance.
| |
LITERATURE CITED |
Ap Rees T
(1994)
Plant physiology: virtue on both sides.
Curr Biol
4:
557-559
[CrossRef][ISI][Medline]
Besford RT
(1990)
The greenhouse effect: acclimation of tomato plants growing in high CO2, relative changes in Calvin cycle enzymes.
J Plant Physiol
136:
458-463
Bevan M
(1984)
Binary Agrobacterium vectors for plant transformation.
Nucleic Acids Res
12:
8711-8721
[Abstract/Free Full Text]
Boorer KJ,
Loo DDF,
Frommer WB,
Wright EM
(1996)
Transport mechanism of the cloned potato H+/sucrose transporter StSUT1.
J Biol Chem
271:
25139-25144
[Abstract/Free Full Text]
Bush DR
(1990)
Electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport.
Plant Physiol
93:
1590-1596
[Abstract/Free Full Text]
Bush DR
(1993)
Proton-coupled sugar and amino acid transporters in plants.
Annu Rev Plant Physiol Plant Mol Biol
44:
513-542
[CrossRef][ISI]
Felsenstein J (1993) PHYLIP (Phylogeny Interference Package),
Version 3.5c. Department of Genetics, University of Washington, Seattle
Frommer WB, Kühn C, Hirner B, Harms K, Martin T, Riesmeier JW,
Schulz B (1996) Sugar transport in higher plants. In M
Smallwood, JP Knox, DJ Bowles, eds, Membranes: Specialized Functions in
Plants. BIOS Scientific Publishers, Oxford, UK, pp 319-335
Gahrtz M,
Schmelzer E,
Stolz J,
Sauer N
(1996)
Expression of the PmSUC1 sucrose carrier gene from Plantago major L. is induced during seed development.
Plant J
9:
93-100
[CrossRef][ISI][Medline]
Gahrtz M,
Stolz J,
Sauer N
(1994)
A phloem-specific sucrose-H+ symporter from Plantago major L. supports the model of apoplastic phloem loading.
Plant J
6:
697-706
[CrossRef][ISI][Medline]
Gamalei Y
(1991)
Phloem loading and its development related to plant evolution from trees to herbs.
Trees
5:
50-64
Hamill JD,
Rounsley S,
Spencer A,
Todd G,
Rhodes MJC
(1991)
The use of the polymerase chain reaction in plant transformation studies.
Plant Cell Rep
10:
221-224
Harms K,
Wöhner RV,
Schulz B,
Frommer WB
(1994)
Expression of plasma membrane H+-ATPase genes in potato.
Plant Mol Biol
26:
979-988
[Medline]
Herbers K,
Meuwly P,
Frommer WB,
Métraux JP,
Sonnewald U
(1996)
Systemic acquired resistance mediated by the ectopic expression of invertase: possible hexose sensing in the secretory pathway.
Plant Cell
8:
793-803
[Abstract]
Hewitt EJ (1966) Sand and Water Culture Methods Used in the Study
of Plant Nutrition, Ed 2. Commonwealth Agricultural Bureau, Technical
Communications No. 22, East Malling, Kent, UK
Hibberd JM,
Whitbread R,
Farrar JF
(1996)
Carbohydrate metabolism in source leaves of barley grown in 700 µmol mol1 CO2 and infected with powdery mildew.
New Phytol
133:
659-671
Hirose T,
Imaizumi N,
Scofield GN,
Furbank RT,
Ohsugi R
(1997)
cDNA cloning and tissue specific expression of a gene for sucrose transporter from rice (Oryza sativa L.).
Plant Cell Physiol
38:
1389-1396
[Abstract/Free Full Text]
Jang JC,
Sheen J
(1994)
Sugar sensing in higher plants.
Plant Cell
6:
1665-1679
[Abstract]
Köster-Töpfer M,
Frommer WB,
Rocha-Sosa M,
Rosahl S,
Schell J,
Willmitzer L
(1989)
A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants.
Mol Gen Genet
219:
390-396
[CrossRef][Medline]
Krapp A,
Quick WP,
Stitt M
(1991)
Ribulose-1,5-bisphosphate carboxylase-oxygenase, other Calvin-cycle enzymes, and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream.
Planta
186:
58-69
Kühn C,
Franceschi VR,
Schulz A,
Lemoine R,
Frommer WB
(1997)
Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements.
Science
275:
1298-1300
[Abstract/Free Full Text]
Kühn C,
Quick WP,
Schulz A,
Sonnewald U,
Frommer WB
(1996)
Companion cell-specific inhibition of the potato sucrose transporter SUT1.
Plant Cell Environ
19:
1115-1123
[CrossRef]
Lemoine R,
Gallet O,
Galliard C,
Frommer WB,
Delrot S
(1992)
Plasma membrane vesicles from source and sink leaves.
Plant Physiol
100:
1150-1156
[Abstract/Free Full Text]
Lemoine R,
Kühn C,
Thiele N,
Delrot S,
Frommer WB
(1996)
Antisense inhibition of the sucrose transporter: effects on amount of carrier and sucrose transport activity.
Plant Cell Environ
19:
1124-1131
[CrossRef]
McCauley MM,
Evert RF
(1989)
Minor veins of the potato (Solanum tuberosum L.) leaf: ultrastructure and plasmodesmatal frequency.
Bot Gaz
150:
351-368
Münch E
(1930)
Die Stoffbewegungen in der Pflanze.
Gustav Fischer, Jena, Germany
Quick WP,
Neuhaus HE,
Stitt M
(1989)
Increased pyrophosphate is responsible for restriction of sucrose synthesis after supplying fluoride to spinach leaf discs.
Biochim Biophys Acta
973:
263-271
Riesmeier JW,
Hirner B,
Frommer WB
(1993)
Potato sucrose transporter expression in minor veins indicates a role in phloem loading.
Plant Cell
5:
1591-1598
[Abstract]
Riesmeier JW,
Willmitzer L,
Frommer WB
(1992)
Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.
EMBO J
11:
4705-4713
[ISI][Medline]
Riesmeier JW,
Willmitzer L,
Frommer WB
(1994)
Antisense repression of the sucrose transporter affects assimilate partitioning in transgenic potato plants.
EMBO J
13:
1-7
[ISI][Medline]
Rocher JP,
Prioul JL
(1987)
Compartmental analysis of assimilate export in a mature maize leaf.
Plant Physiol Biochem
25:
531-540
Sauer N,
Stolz J
(1994)
SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana: expression and characterization in baker's yeast and identification of the histidine-tagged protein.
Plant J
6:
67-77
[CrossRef][ISI][Medline]
Stitt M,
McLilley R,
Gerhardt R,
Heldt HW
(1989)
Metabolic levels in specific cells and subcellular compartments of plant leaves.
Methods Enzymol
174:
518-552
[ISI]
von Schaewen A,
Stitt M,
Schmidt R,
Sonnewald U,
Willmitzer L
(1990)
Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.
EMBO J
9:
3033-3044
[ISI][Medline]
Ward J,
Kühn C,
Tegeder M,
Frommer WB
(1997)
Sucrose transport in plants.
Int Rev Cytol
178:
41-71
Weber H,
Borisjuk L,
Heim U,
Sauer N,
Wobus U
(1997)
A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds.
Plant Cell
9:
895-908
[Abstract/Free Full Text]
Weig A,
Komor E
(1996)
An active sucrose carrier (Scr 1) that is predominantly expressed in the seedling of Ricinus communis L.
J Plant Physiol
147:
685-690
Zhou JJ,
Theodoulou F,
Sauer N,
Sanders D,
Miller AJ
(1997)
A kinetic model with ordered cytoplasmic dissociation for SUC1, an Arabidopsis H+/sucrose cotransporter expressed in Xenopus oocytes.
J Membr Biol
159:
113-125
[CrossRef][ISI][Medline]
Zierler K
(1981)
A critic of compartmental analysis.
Annu Rev Biophys Bioeng
10:
531-562
[CrossRef][ISI][Medline]
Top
Abstract
Introduction
