Research Paper 469

Expression of a Plant Cell Wall Invertase in Roots of Arabidopsis

Leads to Early Flowering and an Increase in Whole Plant Biomass
C. von Schweinichen and M. Büttner
Molekulare Pflanzenphysiologie, Universität Erlangen-Nürnberg, Staudtstraûe 5, 91058 Erlangen, Germany

Received: January 11, 2005; Accepted: May 23, 2005

Abstract: In order to enhance sink strength, we expressed a het- contribute to sink strength. When sucrose is unloaded from
erologous plant cell wall invertase (CrCIN1) under the control of the phloem into the apoplastic space, it can be taken up by sink
a root-specific promoter (ppyk10) in Arabidopsis thaliana. Slightly cells directly or in the form of hexoses after sucrose cleavage
elevated apoplastic invertase activity resulted in apparent phe- by cell wall invertases. Thereby, cell wall invertases create a
notypic changes. Transgenic plants developed more secondary steep sucrose gradient between the phloem and the surround-
roots and subsequently, possibly because of a higher capacity ing sink apoplast which, in turn, very likely drives phloem un-
to acquire nutrients, a higher shoot and whole plant biomass. loading (Eschrich 1989; Miller and Chourey, 1992; Roitsch et
Furthermore, an early flowering phenotype was detected. The al., 1995; Ruan and Patrick, 1995; Weil and Rausch, 1990).
data presented here demonstrate that it is possible to modulate
carbohydrate metabolism by ectopic expression of cell wall in- In Arabidopsis, 17 genes with significant homology to known
vertases and thereby influence sink organ size and whole plant invertases can be found and six of these genes are putative cell
development. wall invertases (Sherson et al., 2003). The expression of two of
these genes, Atbfruct1 (AtcwINV1) and Atbfruct2 (AtcwINV2),
Key words: Arabidopsis, cell wall invertase, sink strength, assim- has been studied in more detail, showing that AtcwINV1 is
ilate partitioning, flowering. expressed in most tissues of mature plants while AtcwINV2
seems to be flower-specific (Tymowska-Lalanne and Kreis,
1998). In addition, RT-PCR analysis (Sherson et al., 2003) re-
vealed distinct expression patterns for AtcwINV3-6. Also in
Introduction other plant species such families of invertase genes exist, as
was shown for maize (Kim et al., 2000), tomato (Godt and
In higher plants, the development and growth of photosyn- Roitsch, 1997; Fridman and Zamir, 2003), potato (Fridman and
thetically inactive sinks is highly dependent on the efficient Zamir, 2003), and for rice (Cho et al., 2005).
and tightly regulated distribution of photoassimilates. Most
plants use sucrose as transport sugar to feed these sinks via Recent data support a pivotal role of invertases in plant sink
the phloem stream (Turgeon, 1989). This source-sink relation- development and growth. The miniature1 mutant of Zea mays,
ship has been investigated using several genetic approaches. which lacks the endosperm-specific cell wall invertase, shows
Transgenic plants that are impaired in the long-distance trans- strongly impaired seed development (Cheng et al., 1996). In
port of sucrose have been generated by inhibiting the phloem Daucus carota, root-specific inhibition of a cell wall invertase
loading step. Drastic effects can be observed in plants when leads to a significant reduction in tap root biomass (Tang et al.,
the sucrose transporter responsible for phloem loading is 1999). During early seed development in Vicia faba, cell wall
down-regulated (Riesmeier et al., 1994; Kühn et al., 1996; Le- invertase activity leads to a high hexose to sucrose ratio (We-
moine, et al., 1996; Bürkle et al., 1998) or knocked out (Gott- ber et al., 1997). This special sugar status seems to promote
wald et al., 2000), including leaf necrosis, inhibition of photo- growth by enhanced cell division. Accordingly, in genotypes
synthesis, delayed flowering and impaired whole plant devel- of V. faba that produce larger seeds, prolonged cell wall inver-
opment due to disturbed assimilate partitioning. Very similar tase activity can be found in their seed coats, and the high hex-
phenotypes can be obtained when a yeast cell wall invertase ose conditions finally lead to an increased cell number in the
is ectopically expressed in plants (von Schaewen et al., 1990; embryo (Weber et al., 1996). In a later phase, when the cell wall
Sonnewald et al., 1991; Heineke et al., 1994; Dickinson et al., invertase is no longer expressed, more sucrose is found in the
1991). During assimilate partitioning, cell wall invertases also apoplast, which can be taken up directly via a sucrose trans-
seem to play an important role in phloem unloading and thus porter to induce the storage phase (Weber et al., 1997). These
data suggest coordinated action of cell wall invertases and
hexose transporters. This model was further supported by the
Plant Biol. 7 (2005): 469 ± 475 coordinated expression of a hexose transporter and a cell wall
 Georg Thieme Verlag KG Stuttgart ´ New York invertase in the host response to powdery mildew infection
DOI 10.1055/s-2005-865894 (Fotopoulos et al., 2003) and by data obtained with Chenopodi-
ISSN 1435-8603 um rubrum suspension cells, where treatment with cytokinin
470 Plant Biology 7 (2005) C. von Schweinichen and M. Büttner

led to the induction of both, invertase and transporter genes primers CrCIN1-5¢ (5¢-CCA TTA TCC ATG GCT TCC TAT AAG TTA
(Ehness and Roitsch, 1997). More recent studies suggesting a CC-3¢), CrCIN1-3¢ (5¢-CAT TCC CAC ATG CCA CTA CG-3¢), AtAct1-
role of cell wall invertases in source/sink regulation mediated 5¢ (5¢-GCG ATG AAG CTC AAT CCA AAC GAG G-3¢), and AtAct1-5¢
by brassinosteroids (Goetz et al., 2000) and demonstrating the (5¢-GGT CAC GAC CAG CAA GAT CAA GAC G-3¢).
requirement of an extracellular invertase for the cytokinin-
mediated delay of senescence (Balibrea Lara et al., 2004) pro- Determination of soluble sugars and starch
vide further insight in the possible mechanism through which
phytohormones and sugars help to regulate plant develop- Soluble sugars and starch were determined in 100 mg of plant
ment. material. The samples were extracted twice with 80 % ethanol
and 20 mM HEPES-KOH, pH 7.5 at 80 8C, and analyzed as de-
In the present study, we wished to modify phloem unloading scribed by Sonnewald (1992).
and sink strength by expressing a plant cell wall invertase un-
der the control of a root-specific promoter. The presented data Determination of invertase activity
indicate that cell wall invertases affect root development and
size and thereby influence physiological processes like flower- Half a gram of plant tissue was homogenized under liquid ni-
ing that might also be regulated by nutrient availability. trogen in five volumes of 25 mM sodium acetate buffer at pH
5.0 containing 0.5 % 2-mercaptoethanol, 10 mM lysine, 1 mM
EDTA, and 0.1 mM phenylmethanesulfonyl fluoride (PMSF).
Materials and Methods
The homogenates were centrifuged at 10 000 ” g for 30 min
Plasmid construction and the supernatants used for determination of the activities
of acid-soluble invertase. The pellets were extensively washed
For expression of CrCIN1 in roots, we cloned the 1452-bp frag- with ice-cold water and cell wall proteins were extracted with
ment C of the pyk10 promoter (EMBL Accession AJ292756) five volumes of 1 M NaCl, containing 1 mM EDTA, overnight at
(Nitz et al., 2001) into the plant transformation vector pBI121 4 8C. All samples were dialyzed overnight at 4 8C against 5 l of
(Clontech) via HindIII and XmaI. Then we amplified the coding 25 mM KPO4 buffer, pH 6.9. lnvertase activity was assayed at
sequence of CrCIN1 (EMBL Accession X81792) from the clone 26 8C on 50 mM sucrose in 13.5 mM citric acid and 26.5 mM
pMB3 (Roitsch et al., 1995) by PCR, introducing a NcoI restric- disodium phosphate at pH 4.6. The glucose concentration was
tion site to the 5¢ end (5¢-CCA TTA TcC ATG GCT TCC TAT AAG determined with Gluco-quant (Roche Diagnostics GmbH).
TTA CC-3¢) and a SacI site to the 3¢ end (5¢-CGG GCG Agc TcG
ATT TTT GAA TGA GC-3¢). The 1747-bp PCR product was cloned
Results
into pBluescript SK+ (Stratagene) and sequenced. A correct
clone was inserted in front of the pyk10 promoter fragment in Expression of the heterologous invertase gene CIN1
pBI121 via XmaI and SacI. coding for a cell wall invertase in roots

Plant transformation and growth In order to investigate the impact of different extracellular
sugar compositions on sink and whole plant development, we
Arabidopsis thaliana plants (ecotype C24) were transformed expressed a cell wall invertase gene under the control of a
with the ªfloral-dip-methodº (Clough and Bent, 1998), using root-specific promoter. To this end, we used the promoter frag-
Agrobacterium tumefaciens strain GV3101. Plants were grown ment C of the pyk10 gene. This truncated promoter was shown
on soil containing 6.5 parts potting soil, 2.5 parts sand, and to be active in whole seedlings, starting 2 days after germina-
1 part lava granules and kept under long-day conditions (16 h tion (DAG) in promoter/GUS reporter plants (Nitz et al., 2001).
light) at 22 8C and 60 % humidity. To grow Arabidopsis hydro- However, GUS staining became more and more root-specific
ponically, we followed the instructions of Gibeaut et al. from 5 DAGs onwards, and was not detectable in aerial parts
(1997). The hydroponic culture was kept under long-day con- after 14 days (Nitz et al., 2001). Therefore, we used a 1452-bp
ditions with 16 h light phase, 22 8C, and 60 % humidity. When fragment (C) of the pyk10 promoter to drive expression of
we first selected transgenic plants on Kanamycin, plants trans- the cell wall invertase gene CIN1 from Chenopodium rubrum
formed with the vector pBI121 alone (vector only transgenic (Fig. 1). Root-specific expression of the CIN1 transgene was
control) were used as controls. However, for the comparative verified by RT-PCR analyses (Figs. 2 A, B). While in wild-type
analyses in the hydroponic system (without Kanamycin selec- plants no CIN1 transcript was detectable, the specific CIN1
tion) we used homozygous CIN1-expressing lines (F2 genera- PCR product could be amplified from roots of transgenic lines
tion) and wild type as a control to avoid possible insertion ef- (Fig. 2 A) but not from aboveground tissues (Fig. 2 B). For quan-
fects of the vector control. For all analyses, wild-type control tification of the CIN1 transcript, gel pictures from three inde-
plants and transgenic plants were grown under identical con- pendent experiments were analyzed using ImageJ software
ditions, both on soil and in the hydroponic system. (http://rsb.info.nih.gov/ij/) and normalization against the cor-
responding Actin controls gave relative CIN1 expression levels
Isolation of RNA and RT-PCR analysis of 1.01  0.16, 0.92  0.14, and 0.77  0.03 for lines ri5, ri7, and
ri10, respectively.
Using the RNeasy mini preparation kit (Qiagen), we isolated
total RNA from 200 mg of plant material. 5 g of RNA were di-
gested with DNase I (Roche) for 40 min at 37 8C before cDNA
was synthesized using the Fermentas RevertAid First Strand
cDNA Synthesis Kit. The PCR reactions were run with Taq poly-
merase, using the AtAct1 gene for internal standardization and
Expression of a Cell Wall Invertase in Roots Affects Flowering and Biomass Plant Biology 7 (2005) 471

Fig. 1 Construct for CIN1 expression in roots.

Organization of the T-DNA region of the plant
transformation vector pBI121 (Clontech) com-
prising the CIN1 coding sequence behind the
1452-bp pyk10 promoter fragment C (Acces-
sion AJ292756). An updated version of the
CIN1 sequence was reported to GenBank (Ac-
cession X81792).

lection) or wild-type plants (without selection) were visible.

Also, when grown on soil until 10 days after germination
(DAG) the transgenic lines were indistinguishable from wild
type. In contrast, from 10 DAG on, ppyk10C::CIN1 plants dis-
played enhanced overall growth with respect to shoot height,
shoot branching, and inflorescence number (Fig. 3 A). Further-
more, while wild-type plants started flowering around 24
DAG, the transgenic lines displayed an early flowering pheno-
type, developing the primary inflorescence 4 to 6 days earlier
than wild type (average 3 weeks after germination). After 4
weeks, ppyk10C::CIN1 plants were taller and displayed more
secondary shoots (Fig. 3 A). To analyze the effects of CIN1 ex-
pression in more detail, we switched to hydroponic growth
conditions (Gibeaut et al., 1997). In this hydroponic system,
we could observe comparable enhanced development of the
CIN1-expressing lines as seen for soil grown plants (Fig. 3 B,
Table 1). In comparison to wild type, transgenic lines devel-
oped shoots and flowers earlier and displayed a higher number
and increased length of secondary shoots with more flowers
and siliques (Table 1).

CIN1-expressing plants exhibit slightly enhanced

cell wall invertase activity in roots

For the further analyses we chose lines ri5, ri7, and ri10, rep-
resenting strong, medium, and weak CIN1-expressing lines
(Fig. 2 A). In the hydroponic system, we determined the total
invertase activity of cell wall extracts from roots and found,
for the transgenic lines, a slight but statistically significant el-
evation of up to 16% (Fig. 4). However, the overall concentra-
tions of soluble sugars (sucrose, glucose, fructose) and starch
Fig. 2 RT-PCR analyses showing CIN1 expression in roots of transgen-
ic lines. (A) A specific 719-bp PCR product can be amplified from trans- in roots of these lines were not altered (data not shown).
genic Arabidopsis lines (ri5: lane 1, ri7: lane 2, ri10: lane 3) expressing
the heterologous invertase gene CIN1 under control of the pyk10 C pro- Expression of CIN1 in roots leads to an increase
moter but not from wild-type plants (lane 4). Actin1 control reactions in root mass as well as whole plant biomass
yielding a specific 391-bp PCR product are shown below each CIN1 re-
action. All reactions were run with 30 (upper panel) and 35 PCR cycles In root cross-sections, the CIN1-expressing lines did not show
(lower panel) to avoid saturating PCR conditions. (B) No CIN1 expres- changes in cell size or cell number (data not shown). However,
sion can be detected in transgenic lines ri5 (lanes 6 and 8) and ri7
transgenic lines differed from wild type in root length and
(lanes 7 and 9), which showed highest CIN1 expression in roots, with
RNA from total aboveground parts (17 DAG; lanes 6 and 7) or from even more pronounced in secondary root number (Fig. 3 B).
inflorescences (29 DAG; lanes 8 and 9). Actin1 control reactions are While in the hydroponic system wild-type plants showed an
shown below each CIN1 reaction. average number of 19.6  2.1 first order secondary roots (mean
value of 8 single plants) with a low degree of higher order side
roots, transgenic ri5, ri7, and ri10 plants developed 27.0  4.6,
Expression of CIN1 in roots leads to early flowering 34.0  3.9, and 28.8  3.7 secondary roots (mean values of 5
single plants each), respectively, with a much higher degree
When we first grew the CIN1-expressing lines on selective me- of higher order side roots. Since these higher order side roots
dium under optimal conditions on plates, no significant differ- were extremely difficult to count, we wished to quantify the
ences to vector only transgenic controls (with Kanamycin se- visible differences in the transgenic lines by determining root
472 Plant Biology 7 (2005) C. von Schweinichen and M. Büttner

Fig. 3 Phenotypic changes of transgenic CIN1-expressing lines. (A) pared to wild-type control plants. (B) Four-week-old transgenic lines
Four-week-old transgenic lines (ri3, ri5, ri7, ri10) expressing the heter- (ri5, ri7) grown hydroponically, showing comparable increase in shoot
ologous invertase gene CIN1 under the control of the pyk10C-promoter branching and early flowering. In addition, enhanced root growth and
grown on soil, showing enhanced development of above-ground parts root branching can be seen.
including increased shoot branching and early flowering when com-

Fig. 5 Biomass determination of roots, shoots, and whole plants.

Transgenic ppyk10C::CIN1 lines (ri5, ri7, ri10) were grown hydroponical-
ly and analyzed for their fresh weight of roots, shoots, and whole
plants. Data represent mean values of ten independent measurements
Fig. 4 Total invertase activity of cell wall extracts from roots. The (bars represent the standard error SE). Transgenic lines have 20 ± 60%
ppyk10C::CIN1 lines (ri5, ri7, ri10) were grown hydroponically and ana- more biomass than the wild type.
lyzed for apoplastic invertase activity using cell wall extracts from
roots. Data represent mean values of three independent measure-
ments (bars represent the standard error SE). Transgenic lines show
slightly enhanced invertase activity over the wild-type control. (Table 1). The relative gain in fresh weight in the analyzed
transgenic lines corresponds with the CIN1 expression levels
determined by semi-quantitative RT-PCR (Fig. 2 A), indicating
a direct effect of invertase overexpression and root develop-
mass. We measured a clear increase in fresh weight of up to ment. The same increase could be measured after freeze-dry-
60 % as compared to wild-type plants (Fig. 5). Interestingly, ing the root or whole plant material and measuring the dry
not only the root weight was significantly higher but also the weight (data not shown), indicating that the increase in root
shoot fresh weight showed a similar increase (Fig. 5), result- and whole plant mass is not merely due to elevated water con-
ing from an increase in secondary shoot number and height tent but reflects increased biomass.
Expression of a Cell Wall Invertase in Roots Affects Flowering and Biomass Plant Biology 7 (2005) 473

Table 1 Enhanced development of transgenic Arabidopsis plants expressing the cell wall invertase CIN1 gene under control of the pyk10C pro-
moter in comparison to wild-type plants. Mean values  standard errors from at least nine independent plants per line are shown. Probability val-
ues as determined by Students t-tests are indicated by asterisks: * p < 0.05, ** p < 0.01

Line 21 DAG 24 DAG 26 DAG 27 DAG 28 DAG 29 DAG

Number of WT 0.25  0.13 0.83  0.11 2.42  0.63 4.00  0.77 6.08  0.92 7.08  1.04
shoots ri2 0.75  0.16* 2.63  0.75* 6.38  1.53* 8.00  1.70* 9.88  1.44* 10.88  1.19*
ri3 0.56  0.18 1.78  0.64 5.44  1.28* 7.56  1.31* 9.56  1.02* 11.33  0.76**
ri5 0.56  0.18 1.89  0.54 5.11  0.82** 7.44  1.02* 9.89  0.82** 11.33  1.01*
ri7 0.70  0.21 2.70  0.78* 6.00  1.03** 7.30  1.19* 8.70  1.18 10.30  1.18
ri10 0.78  0.32 2.56  1.08 3.67  1.15 5.00  1.46 6.67  1.60 7.67  1.76

Shoot length WT 0.42  0.26 4.08  1.59 24.75  7.88 47.08  12.84 79.00  17.30 112.42  18.80
(mm) ri2 8.63  4.31 45.63  17.26* 104.00  26.34* 134.63  28.43* 169.38  28.90* 197.13  26.77*
ri3 1.44  0.71 16.89  6.69 66.44  17.60* 103.00  20.27* 147.33  20.70* 189.11  19.58**
ri5 4.33  2.71 29.56  11.81 76.44  17.98* 116.11  18.66** 157.44  17.85** 187.56  15.77**
ri7 8.70  4.35 50.10  18.33 104.30  27.34* 138.00  31.10* 167.80  31.88* 196.80  30.36*
ri10 6.78  5.69 23.89  15.55 53.11  23.06 74.67  26.49 100.11  30.53 123.44  32.65

Total number WT 0.00  0.00 0.00  0.00 0.33  0.26 0.92  0.48 2.42  0.91 4.58  1.42
of flowers ri2 0.00  0.00 1.13  0.64 4.00  1.61 8.13  3.29 13.25  5.02 22.63  7.36*
ri3 0.00  0.00 0.22  0.22 1.44  0.99 3.33  1.64 7.89  3.17 17.11  6.43
ri5 0.00  0.00 0.78  0.46 2.67  1.19 4.89  1.80 11.33  3.31* 18.67  5.26*
ri7 0.00  0.00 1.00  0.63 4.90  1.82 * 8.60  3.43* 16.80  5.82* 27.40  8.62*
ri10 0.22  0.22 1.00  0.88 4.11  3.20 6.89  4.64 11.00  6.59 15.78  8.93

Total number WT 0.00  0.00 0.00  0.00 0.00  0.00 0.08  0.08 0.67  0.36 1.33  0.54
of siliques ri2 0.00  0.00 0.50  0.38 1.75  0.90 3.00  1.32 5.75  2.14* 10.38  4.06
ri3 0.00  0.00 0.00  0.00 0.33  0.33 0.67  0.47 2.11  1.40 4.56  1.96
ri5 0.00  0.00 0.22  0.22 0.89  0.51 1.89  0.93 3.78  1.53 6.33  2.14*
ri7 0.00  0.00 0.20  0.20 2.00  0.94 3.80  1.43* 6.60  2.41* 12.10  4.95
ri10 0.00  0.00 0.57  0.57 2.43  1.97 4.43  3.66 6.29  4.62 10.29  6.78

Discussion vealed only limited sequence conservation between invertase

inhibitors from different plant species (Rausch and Greiner,
Cell wall invertases seem to play an important role in deter- 2004).
mining sink strength by increasing the sucrose gradient be-
tween the phloem and the sink apoplast. In order to modu- When we examined the ppyk10C::CIN1 plants in detail, we
late assimilate partitioning and to enhance sink strength we first found changes in flowering time. Flowering started 4 to
expressed a plant cell wall invertase under the control of the 6 days earlier than in wild-type control plants and a higher
pyk10C promoter fragment. This truncated promoter drives inflorescence number developed due to more secondary
a characteristic pattern of GUS expression in transgenic Ara- shoots. A similar phenotype was observed when a yeast cell
bidopsis plants during plant development, resulting in root wall invertase gene (ScSUC2) was expressed under control of
specificity in advanced developmental stages (Nitz et al., the meristem-specific KnAT1 gene promoter (Heyer et al.,
2001). When we used this truncated promoter to drive CIN1 2004). The pKnAT1::ScSUC2 plants develop more siliques and,
expression, transgenic ppyk10C::CIN1 plants showed apparent in turn, a higher seed yield resulting from enhanced branching
changes with respect to their flowering time, morphology, and of the secondary shoots (axillary inflorescences). While the
biomass. Most studies addressing related questions so far have ppyk10C::CIN1 plants in our study did not show enhanced
used a yeast cell wall invertase with a signal peptide for apo- branching of secondary shoots, they developed a higher num-
plastic targeting (von Schaewen et al., 1990; Sonnewald et al., ber of secondary shoots and thus more siliques (Table 1).
1991; Bussis et al., 1997; Sonnewald et al., 1997; Weber et al.,
1998; Neubohn et al., 2000; Zuther et al., 2004; Heyer et al., As an additional phenotypic change in the ppyk10C::CIN1
2004). However, when we expressed the same yeast cell wall plants, we observed an increase in root fresh and dry weight
invertase (kindly provided by Prof. U. Sonnewald) under con- resulting from longer roots and a higher number of secondary
trol of the pyk10C promoter, we could not observe any effects roots. Furthermore, transgenic plants developed more and
comparable with those found for the ppyk10C::CIN1 plants, as faster-growing secondary shoots leading to a higher fresh
described below. Furthermore, we used a heterologous en- weight as compared to wild-type control plants. These effects
zyme from a different plant species (CIN1 from C. rubrum) to on aerial parts, where the pyk10C promoter is not active, may
circumvent possible effects of endogenous Arabidopsis inver- be more indirect. Possibly, the increase in root mass, and con-
tase inhibitors (Greiner et al., 1998), since recent studies re- sequently in root surface, allows more effective absorption of
474 Plant Biology 7 (2005) C. von Schweinichen and M. Büttner

nutrients from the medium, resulting in enhanced overall de- for normal development of endosperm and maternal cells in the
velopment. pedicel. Plant Cell 8, 971 ± 983.
Cho, J. I., Lee, S. K., Ko, S., Kim, H. K., Jun, S. H., Lee, Y. H., Bhoo, S. H.,
Although we found a slight increase in total cell wall invertase Lee, K. W., An, G., Hahn, T. R., and Jeon, J. S. (2005) Molecular clon-
activity, we could not measure significant differences in the ing and expression analysis of the cell-wall invertase gene family
concentrations of soluble sugars in roots of the transgenic in rice (Oryza sativa L.). Plant Cell Reports 24, 225 ± 236.
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for
plants in comparison to wild type, suggesting that the ob-
Agrobacterium-mediated transformation of Arabidopsis thaliana.
served phenotypes do not result from changes in the steady-
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Gibeaut, D. M., Hulett, J., Cramer, G. R., and Seemann, J. R. (1997)
ratio of soluble sugars might lead to a short temporal or local Maximal biomass of Arabidopsis thaliana using a simple, low-
increase in the apoplastic hexose concentration, resulting in maintenance hydroponic method and favorable environmental
higher cell division activity and, in turn, to enhanced root conditions. Plant Physiology 115, 317 ± 319.
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found in large-seeded V. faba genotypes, where prolonged growth phenotype of transgenic tomato expressing apoplasmic in-
cell wall invertase activity in the seed coat and the resulting vertase. Plant Physiology 95, 420 ± 425.
high hexose conditions, finally lead to an increased cell num- Ehness, R. and Roitsch, T. (1997) Co-ordinated induction of mRNAs
ber in the embryo (Weber et al., 1996). This idea is further sup- for extracellular invertase and a glucose transporter in Chenopodi-
ported by the finding that CIN1 expression also shows similar um rubrum by cytokinins. The Plant Journal 11, 539 ± 548.
effects when driven by a different sink-specific promoter (von Eschrich, W. (1989) Phloem unloading of photoassimilates. In Trans-
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promoter did not further affect the observed phenotypes of
fruct1, are induced in Arabidopsis during infection with the fungal
the ppyk10C::CIN1 plants (data not shown), suggesting that in
biotroph Erysiphe cichoracearum. Plant Physiology 132, 821 ± 829.
roots such a transporter is constitutively expressed and that
Fridman, E. and Zamir, D. (2003) Functional divergence of a syntenic
root cells always have a high capacity to take up hexoses. Such invertase gene family in tomato, potato, and Arabidopsis. Plant
AtSTPs have already been identified in Arabidopsis roots Physiology 131, 603 ± 609.
(AtSTP1: Sauer et al., 1990; Sherson et al., 2000; AtSTP4: Truer- Godt, D. E. and Roitsch, T. (1997). Regulation and tissue-specific dis-
nit et al., 1996; AtSTP13: Büttner, unpublished). tribution of mRNAs for three extracellular invertase isoenzymes of
tomato suggests an important function in establishing and main-
Taken together, the presented data suggest that the sink ca- taining sink metabolism. Plant Physiology 115, 273 ± 282.
pacity to take up monosaccharides is not limited and that the Goetz, M., Godt, D. E., and Roitsch, T. (2000) Tissue-specific induction
decision whether sinks are provided with sucrose or hexoses of the mRNA for an extracellular invertase isoenzyme of tomato by
is regulated by the activity of cell wall invertases. Therefore, it brassinosteroids suggests a role for steroid hormones in assimilate
is possible to modulate carbohydrate metabolism by ectopic partitioning. The Plant Journal 22, 515 ± 522.
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Acknowledgements apoplasmic invertase inhibitor. Proof of function of the recombi-
nant protein and expression analysis during plant development.
This work was supported by the Deutsche Forschungsgemein-
Plant Physiology 116, 733 ± 742.
schaft (grant Bu 973/3). We thank Norbert Sauer for his contin- Heineke, D., Wildenberger, K., Sonnewald, U., Willmitzer, L., and
uous support and for helpful discussions. We also thank Prof. Heldt, H. W. (1994) Accumulation of hexoses in leaf vacuoles:
T. Roitsch for the CrCIN1 cDNA clone and Prof. F. Grundler for Studies with transgenic tobacco plants expressing yeast-derived
the pyk10 promoter clone. invertase in the cytosol, vacuole or apoplasm. Planta 194, 29 ± 33.
Heyer, A. G., Raap, M., Schroeer, B., Marty, B., and Willmitzer, L. (2004)
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