Advances in Bioscience and Biotechnology, 2010, 1, 426-438 ABB
doi:10.4236/abb.2010.15056 Published Online December 2010 (
Published Online December 2010 in SciRes.
Overexpression of aspen sucrose synthase gene promotes
growth and development of transgenic Arabidopsis plants
Fuyu Xu1, Chandrashekhar P. Joshi1, 2
1Biotechnology Research Center School of Forest Resources and Environmental Science, Michigan Technological University,
Houghton, USA;
2Department of Bioenergy Science and Technology, Chonnam National University, Buk-Gu, Gwangju, Korea.
Received 14 September 2010; revised 22 October 2010; accepted 29 October 2010.
In plants, sucrose synthase (SUS) enzymes catalyze
conversion of sucrose into fructose and UDP-glucose
in the presence of UDP. To investigate the impact of
overexpression of heterologous SUS on the growth
and development of Arabidopsis, we transformed
Arabidopsis plants with an overexpression vector
containing an aspen SUS gene (PtrSUS1). The ge-
nomic PCR confirmed the successful integration of
PtrSUS1 transgene in the Arabidopsis genome.
PtrSUS1 expression in transgenic Arabidopsis plants
was confirmed by RT-PCR. The SUS activity was
dramatically increased in all transgenic lines exam-
ined. The three selected transgenic PtrSUS1 lines ex-
hibited faster growth and flowered about 10 days
earlier compared to untransformed controls, and also
possessed 133%, 139%, and 143% SUS activity
compared to controls. Both fresh weights and dry
biomass yields of the whole plants from these three
selected transgenic lines were significantly increased
to 125% of the controls. Transgenic PtrSUS1 lines
also had a higher tolerance to higher concentration of
sucrose which was reflective of the increased SUS
activity in transgenic versus wild-type plants. The
growth differences between wild-type and transgenic
plants, either in root and hypocotyl length or in fresh
and dry weight of whole plant, became more pro-
nounced on the media containing higher sucrose
concentrations. Taken together, these results showed
that the early flowering, faster growth and increased
tolerance to higher sucrose in transgenic lines were
caused by the genome integration and constitutive
expression of the aspen PtrSUS1 gene in transgenic
Keywords: Arabidopsis; Aspen Trees (Populus
Tremuloides); Over-Expression; Sucrose Synthase
(SUS); SUS Enzyme Activity
Sucrose synthase (SUS) is an important enzyme in car-
bohydrate metabolism of plants that catalyzes a reverse-
ble reaction of converting sucrose into fructose and
UDP-glucose in the presence of UDP. SUS, therefore,
mainly functions in tissues that actively metabolize su-
crose [1-3]. The level and distribution of SUS activity in
different tissues provides a meaningful insight into the
transport and consumption of carbohydrates during the
plant development and plant response to various envi-
ronmental stresses [4,5]. SUS activity has been localized
in different intracellular structures, such as cell mem-
branes [6,7], the cytoskeleton [8], and the tonoplast [9].
SUS also exists in two forms, the soluble form in the
cytosol and the second form in association with the
plasma membrane or cell walls [10,11], with the latter
proposed to be involved in the synthesis of cell wall
components by providing UDP-glucose directly to the
cellulose synthases and callose synthases [12]. Using
differentiating tracheary elements as a model system,
Salnikov et al. [13] and Salnikov et al. [14] showed that
the SUS was specifically localized in and around the
plasma membranes and the microtubules during the
secondary wall thickenings, and thus establishing a
spatial relationship and context with secondary wall
cellulose synthesis. SUS enzyme activity was localized
in roots of wheat in response to hypoxic conditions, and
was linked with the secondary wall thickenings and the
deposition pattern of cellulose [15,16]. SUS activity has
also been positively linked with sink strength in storage
organs of potatoes [17], tomato fruits [18], developing
cotton seed [19], maize kernels [20,21], and pea em-
bryos [22,23].
The involvement of SUS in plant growth and repro-
ductive development was investigated by several re-
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
search groups. The over-expression of cotton SUS in
tobacco transgenic plants increased the total biomass
amount [24]. While studying the activities of enzymes
involved in the control of carbon flux of two genotypes
of Vigna radiata (mung bean) differing in seed weight
and size, Chopra et al. [25] found that the change of
SUS activities was positively correlated with the bio-
mass accumulation of seeds in two genotypes, i.e., the
large-seed genotype maintained high SUS activity longer,
thus with the longer seed filling phase and stronger sink
compared to the small seeds. It has also been reported
that SUS cooperates with UGPase and AGPase to con-
trol the efficient partitioning of sucrose into ADP glu-
cose and thereby regulate the seed sink strength in the
mung bean plants [26,27]. During the flower bud forma-
tion, greater activities of SUS and other sugar cataboliz-
ing enzymes may enhance the capacity of buds to attract
assimilates, thus accelerating bud growth and increasing
the number of bud primordia [28]. It has recently been
reported that AtSUS from Arabidopsis showed specific
roles in seed and silique development [5]. Overexpres-
sion of the cotton SUS gene in hybrid poplar promoted
2~6% of cellulose content and increased crystallinity in
all transgenic lines with the increased SUS enzyme ac-
tivity compared to controls [29]. However, the overex-
pression of the same cotton SUS construct into tobacco
did not change the cellulose percentage [24]. The differ-
ence between transgenic tobacco and transgenic hybrid
poplar plants is probably due to the less secondary
growth in tobacco compared to poplars.
The role of SUS in flowering process has also been
examined using antisense SUS constructs in transgenic
tomato (Lycopersicon esculentum) plants, in which SUS
activity was severely inhibited by the antisense RNA in
flowers and fruit pericarp tissues, and in turn causing a
reduced fruit set and a slower growth rate of fruits [30].
The dramatically decreased sucrose unloading capacity
of 7-day-old fruit was observed in antisense lines with
low SUS activity, accompanied by a slower growth rate
of antisense fruit from the first week of flowering and a
reduced fruit set. These results suggest that SUS partici-
pates in regulating sucrose import capacity of young
tomato fruit, which is a determinant for fruit set and de-
velopment. SUS gene expression was dramatically re-
duced in transgenic cotton plants expressing a SUS co-
suppression construct, which caused the reduced number
of fiber initiations and reduced fiber length, and induced
numerous collapsed fiber cells, and even resulted in the
failure of setting of seeds in some lines [31]. These re-
sults also supported a specific role for SUS during fiber
initiation and elongation in cotton.
The release of poplar genome and availability of pop-
lar EST/cDNA libraries provided excellent platform to
investigate the function of SUS family in plant growth
and development. The results of genome-wide transcript
profiling have shown that SUS1 and SUS2 were the most
abundant transcripts among poplar SUS family members
(Xu and Joshi, submitted). In addition, PttSUS1 and
PttSUS2 from Populus tremula (L.) × tremuloides
(Michx.) were also shown to dramatically increase dur-
ing tension wood (TW) formation, supporting the sig-
nificant role of SUS in cellulose synthesis in TW [32].
Previously, our lab successfully cloned PtrSUS1 and
PtrSUS2 from aspen trees (Populus tremuloides Michx.),
and we also found that they were up-regulated in devel-
oping xylem of TW (Joshi et al., unpublished data). So
we were interested to investigate the effects of over-
expression of heterologous PtrSUS in Arabidopsis. In
this study, we show the successful integration of aspen
SUS gene (PtrSUS1) in Arabidopsis genome by genomic
PCR, overexpression of PtrSUS1 genes in Arabidopsis
through RT-PCR, and increased SUS enzyme activities
in transgenic plants, suggesting that the observed faster
growth during early developmental stages and early
flowering in transgenic lines were caused by the integra-
tion and overexpression of the aspen SUS gene in the
Arabidopsis genome.
2.1. Plant Materials
Arabidopsis thaliana wild-type (Col-0), SUS overex-
pressing transgenic lines and pBI121 (containing
35S-GUS-NOS construct) transgenic control lines were
grown at 22 under 16 h:8 h light:dark conditions with
approximately 200 μm photons m2 sec1 light intensity
either on Arabidopsis soil mix (Lehle Seeds, Round
Rock, TX, USA) or agar plates.
2.2. DNA Constructs and Plant Transformation
The PtrSUS1 overexpression construct was prepared by
Drs. Takeshi Fujino and Suchita Bhandari in our labora-
tory. PCR was performed with the following primers
using a full-length PtrSUS1 cDNA of 2.84 kb (GenBank
accession#: AY341026) as a template. The forward
primer was Susy_F_SpeI (5’-CGACTAGTGGCAT-
TAAACTTAAGGAGC-3’); the reverse primer was
TTCC- 3’). The PCR product was first cloned in PCR2.1
Topo vector (Invitrogen) and digested with SpeI and
SmaI, and the insert was cloned into the binary plant
vector pBI121 (Clontech, San Diego, CA, USA) di-
gested with XbaI (SpeI compatible) and Ecl136II (blunt),
between the cauliflower mosaic virus 35S promoter
(35SP) and Nopaline synthase terminator (NOST). The
resultant plasmid, named 35S-PtrSuS1-NOST, was mo-
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
bilized to Agrobacterium tumefaciens strain C58 and
used for plant transformation. Arabidopsis thaliana
wild-type (Col-0) plants were transformed with the con-
struct by the Agrobacterium-mediated floral dip method
[33], and transformed seeds were surface-sterilized,
sown and selected on 0.5x MS-medium containing 1%
(w/v) sucrose and 100 µg ml-1 kanamycin in Petri dishes.
Transgenic seeds were germinated and grown in tissue
culture room at 24 ± 2 under a 16-h light/8-h dark
illumination regime with about 150 µmol m2 sec1
fluorescent illumination in daylight setting. Kanamy-
cin-resistant seedlings (T1) were transplanted into soil
and grown to maturity for seed harvesting. T2 seeds were
collected from selfed T1 plants, and a portion of the T2
seeds was germinated on 0.5x MS medium with kana-
mycin selection and transplanted into soil. T3 seeds were
harvested from T2 plants and used in this study.
2.3. PCR Confirmation of PtrSUS1 Transgene
Integration in Arabidopsis
Genomic DNA was extracted from the leaves of trans-
genic Arabidopsis seedlings of T1, T2, and T3 generations
grown in soil after selection on 0.5x MS medium con-
taining Kanamycin. Twelve independent transgenic lines
were produced and confirmed in this study. Transgenic
nature of selected lines was confirmed by three pairs of
primers: 1st pair containing a forward primer from 35S
promoter sequence (35SP-F) and a reverse internal
primer from PtrSUS1 (SusyInR); 2nd pair containing an
internal forward primer derived from PtrSUS1 (SusyInF)
and a reverse primer (NostR) from the NOST; 3rd pair
containing SusyInF and SusyInR primers.
2.4. Growth of Transgenic Arabidopsis Lines on
Media with Varying Sucrose Concentrations
Sterilized seeds of wild type and T3 transgenic Arabi-
dopsis lines were cultured on 0.5x MS medium without
sucrose in Petri dishes, vertically placed in racks, for 3
days, and were transplanted onto 0.5x MS medium with
0, 1, 2, 3, 4, 6, 8% sucrose to grow either in regular light
condition or wrapped with aluminum foil for dark treat-
ment. Each plate contained 15 wild-type or PtrSUS1
transgenic plants. Root and hypocotyl lengths were
measured on day 7 day after the transfer. Average length
was taken from one set of 10 plants and three replicate
plates per treatment were used for each transgenic line.
The fresh weight of each individual whole plant was
measured immediately after the harvest. Biomass yield
(dry weight in mg) was recorded after drying in an oven
to practically zero per cent moisture content roughly
after 48 h at 70. Whole plants were harvested for
biomass measurements and total protein extraction after
3 weeks of transfer.
2.5. RNA Preparation and Semi-Quantitative
Seedlings were harvested after different periods of incu-
bation on serial concentrations of sucrose. Total RNA
was isolated by using RNeasy Plant Mini Kit (Qiagen,
Valencia, CA, USA), and treated with DNase I (NEB,
MA, USA). PtrSUS1 transcripts in transgenic Arabidop-
sis plants were determined by RT-PCR. 1 µg of total
RNA was used as a template for first-strand cDNA syn-
thesis with SuperScript® II RT kit (Invitrogen). The
synthesis of the first strand cDNA was conducted at
42 for 30 min followed by 70 for 10 min. 2 µl of RT
reaction (cDNA) which is equivalent to 100 ng of RNA
was used in the reaction of 25 µl with GoTaq® Green
PCR Master Mix (Promega, Madison, WI). In order to
differentiate PtrSUS1 expression from the endogenous
AtSUS gene expression, we designed PtrSUS1 gene-
specific primer pairs using six AtSUS cDNA sequences
as references while using PRIMER3 software (http:// Pri-
mers were purchased from Sigma (St. Louis, MO). The
following primers were used: for PtrSUS1 gene,
for ubiquitin gene (At4g05320), AtUBQ-F (5’-GAA
tion was conducted at 94 for 10 min, then 40 cycles of
94 for 30 sec, 60 for 30 sec, and 72 for 1 min,
followed by an additional extension of 72 10 min.
2.6. In Situ Staining of Sucrose Synthase Activity
Arabidopsis seedlings were prepared according to Ser-
geeva et al. [34] with some modifications. Briefly, soaked
seeds were grown in Petri dishes with two layers of
moist filter paper and placed at 4 for stratification in
darkness for 2 days. They were then transferred to 23
in the light with a 16h:8h day:night regime and grown
for 7 d. Roots from poplar were obtained from tissue
culture as described above. In situ staining and localiza-
tion of sucrose synthase activity were conducted by in-
cubating whole plants or roots in a reaction medium as
described earlier [34-36] with some modifications. The
incubation medium contained 100 mM HEPES-NaOH
buffer (pH 7.4), 10 mM MgCl2, 2 mM EDTA, 0.2% BSA,
2 mM EGTA, 1 mM NAD, 1 U phosphoglucomutase
(PGM) from rabbit muscle, 1 U glucosephosphate dehy-
drogenase (G6PDH) from Leuconostoc mesenteroides,
20 µM glucose-1,6-bisphosphate , 1 U UDPG-pyrophos-
phorylase (UGPase) from beef liver (Sigma, St. Louis,
MO), and 0.03% NBT, and the reaction was started by
adding substrate solution (containing sucrose, UDP and
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
PPi) resulting in final concentration of substrates in the
incubation medium of 3.6 mM, 71 µM, and 71 µM, re-
spectively. Controls were incubated without sucrose, or
without PGM, glucose-1,6-bisphosphate and PPi, or
without NAD.
2.7. Quantitative SUS Enzyme Assays
A 0.5-1.0 g sample of leaves or whole plants was
grounded in liquid nitrogen with a mortar and pestle.
Extraction buffer contained 50 mm HEPES-KOH (pH
7.4), 1 mm MgCl2, 1 mm EDTA, 1 mM EGTA, 10% (v/v)
glycerol, 0.1% (w/v) bovine serum albumin (BSA), 5
mM DTT, 20 mM β-mercaptoethanol, 1% (w/v) insolu-
ble PVP-40 (polyvinylpyrrolidone), and 0.1 mM PMSF
(phenylmethylsulfonyl fluoride), 1 µg/ml pepstatin, and
2 µg/ml leupeptin. The ratio of powdered tissue to ex-
traction buffer was about 5 ml/g fresh weight. The ho-
mogenate was centrifuged at 4 at 13,000 g for 5 min.
The supernatant was collected and equilibrated with 50
mm Hepes-NaOH (pH 7.5), 1 mm EDTA and 1 mm DTT.
Protein extract concentrations were determined using a
Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) with
BSA as the standard.
Quantitative sucrose enzyme activity assays were car-
ried out in the direction of sucrose cleavage according to
Xu et al. [35], Zrenner et al. [17] and Ruan et al. [31]
with minor modifications, and UDP-glucose from su-
crose was measured spectrophotometrically by the ab-
sorption change at 340 nm molar extinction (A340) as
NAD was reduced to NADH by UDP-Glc dehydrogenase
(UDPG-DH; Sigma, St. Louis, Mo.). Briefly, each assay
was carried out with 30-40 μl of crude extract equiva-
lent to about 40 μg total protein in 250 μl reaction mix-
tures containing 20 mM HEPES-KOH (pH 7.0), 100
mM sucrose and 4 mM UDP. A blank without sucrose
and UDP was included to subtract the background. In-
cubation was carried out at 25 for 40 min and stopped
by boiling for 5 min. The determination of UDP-glucose
was conducted in 200 mM glycine-NaOH (pH 8.9), 5
mm MgCl2, 2 mm NAD and 0.04 units of UDP-glucose
dehydrogenase, and the reaction mixture was incubated
for 30 min at 37 before measurement of A340. Blanks
without sucrose and UDP were included to subtract the
3.1. Generation and Confirmation of PtrSUS1
Transgenic Arabidopsis Plants
PtrSUS1, a full-length aspen SUS cDNA (GenBank ac-
cession # AY341026) was successfully cloned into the
binary vector pBI121 replacing the GUS gene (Figure 1).
Agrobacterium-mediated floral dip transformation me-
thod was used to produce transformed seeds. Kanamy-
cin-resistant plants were subjected to genomic PCR con-
firmation. Three pairs of primers were used to verify
integration of the PtrSUS1 transgene in Arabidopsis as
described in Materials and methods and all tested trans-
genic lines were found to carry the transgene. Figure 2(a)
shows the results from one of the representative lines,
and the successful transgenic events with intact PtrSUS1
cDNA integration show three fragments: ~2 kb from
35SP-F::SusyInR, ~1.2 kb from SusyInF::NostR, and
366 bp SusyInF::SusyInR primer pairs as expected.
Three representative transgenic Arabidopsis lines, de-
noted as T3-6, T3-7, and T3-11 (Figure 2(c)), were se-
lected for further analyses.
Under standard growth conditions in the greenhouse,
transgenic lines were phenotypically indistinguishable
from the wild type plants before inflorescence emer-
gence. However, the speed of inflorescence development
and timing of flowering from PtrSUS1 transgenic plants
were much faster and earlier than wild-type plants (Fig-
ure 2(b) and Table 1).
3.2. Overexpression of PtrSUS1 Transgene in
Semi-quantitative RT-PCR was performed to confirm the
expression of the PtrSUS1 transgene in T3 plants from
three homozygous transgenic lines. The total RNA was
extracted from leaves of three transgenic lines and cor-
responding controls grown in soil and/or cultured in
Petri dishes. Expression of an Arabidopsis ubiquitin
gene was used as an internal control. Using PtrSUS1
gene-specific primers that can discriminate between as-
Figure 1. Structure of the 35S-PtrSUS1-NOST construct used
for Arabidopsis transformation. To introduce the full-length
PtrSUS1 cDNA into Arabidopsis genome by Agrobacterium-
mediated method, the XbaI–Ecl136II fragment of PtrSUS1 was
inserted between the cauliflower mosaic virus 35S promoter
(35SP) and the nopaline synthase gene terminator (NOST) in a
binary plant vector pBI121, named 35S-PtrSUS1-NOST. pBI121
carries the neomycin phosphotransferase II (NPTII) gene,
driven by the nopaline synthase gene promoter (NOSP), for
selection of transgenic cells by kanamycin.
Table 1. Time of flowering for wild type and transgenic plants.
Genotype Col-0 T3-6 T3-7 T3-11
Days of first
flower buds visible 23 15 13 14
Note: Days, averaging day from date of sowing seeds in Petri dish, exclude-
ing a 3-day stratification at 4.
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
(a) (b)
T3-6 T3-7 T3-11 Col-0
Figure 2. Genomic PCR confirmation and phenotypes of rep-
resentative PtrSUS1 transgenic Arabidopsis. Three primer
pairs were used to verify the PtrSUS1 transgenic Arabidopsis
plants (a); 15 d-old seedlings from transgenic plants of one line
and wild-type (Col-0) (b); Growth of three representative
transgenic lines and wild-type in 0.5x MS medium containing
100 µg/ml kanamycin.
pen and Arabidopsis SUS genes, all three transgenic
lines tested showed expression of the PtrSUS1 gene but
it was absent in other control plants (Figure 3). Similar
results were obtained by using plants grown in green-
house and Petri dish samples since the PtrSUS1 gene is
under the control of a constitutive 35S CaMV promoter.
3.3. SUS Enzyme Activity
We tested whether SUS enzyme activity was enhanced
in transgenic lines using in situ staining, as was de-
scribed previously for tissue sections [36,37]. Whole
seedlings were fixed and incubated in the staining reac-
tion buffer. Figure 4 shows the results from the roots of
the wild type and transgenic plants but similar results
were obtained using other tissues. Blue formazan color
from reduced nitroblue tetrazolium (NBT) was observed,
indicating enzyme activity. This blue precipitation spe-
cifically indicated SUS activity, as compared and deter-
mined from the absence of staining in the control reac-
tions without sucrose. Transgenic lines showed stronger
blue color, indicating stronger SUS enzymatic activity
compared to wild-type untransformed controls (Figure
Protein extracts of transgenic and wild type plants
were further assayed for quantitative SUS enzyme ac-
tivities by determining the rate of UDP-glucose produc-
tion. SUS activities of three transgenic plants were 480,
501, and 518 nmol UDP-glucose per minute and per mg
of protein, corresponding to 133%, 139%, and 143% of
wild type (361 nmol UDP-glucose per minute and mg
protein) (Figure 5), and the difference between trans-
genic and wild type plants was statistically very highly
or highly significant. This demonstrated that introduce-
tion of aspen SUS gene PtrSUS1 resulted in significant
increase of SUS activity in transgenic Arabidopsis
Figure 3. Overexpression of the PtrSUS1 gene in transgenic
plants. Three-week-old seedlings of the wild-type (Col-0),
GUS vector carrying control plants (pBI121: p35S-GUS) and
three lines of transgenic plants overexpressing PtrSUS1
(p35S-PtrSUS1) were collected from both greenhouse and
vertical Petri dishes were used for total RNA isolation and
subsequent RT-PCR. Expression of an Arabidopsis ubiquitin
gene was used as an internal control. PtrSUS1 Gene-specific
primers as described in “Materials and methods” were used to
determine the presence and level of PtrSUS1 transcript of
transgenic plants.
Figure 4. Sucrose synthase activity in 7-d-old roots of WT
(Col-0) and three transgenic lines T3-6, T3-7 and T3-11.
Stained roots from whole plant staining including one technical
reaction control without sucrose.
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
Figure 5. Sucrose synthase (SUS) activity in wild-type and three PtrSUS1 transgenic lines of
Arabidopsis thaliana. SUS activity measured from the pooled young leaves and shoots of 18-d-old
Arabidopsis grown in greenhouse. SUS activity was higher in transgenic plants. Error bars indi-
cate the standard error (SE: n = 3). Two asterisks **indicate very significant difference at the 99%
level; three asterisks ***indicate highly significant difference at the 99.9% level.
By using both the methods, histochemical observa-
tions and biochemical measurements, we have obtained
consistent results and have demonstrated the increase in
SUS enzyme activity in the transgenic plants.
3.4. Biomass Accumulation
We further examined whether transgenic plants accumu-
lated more biomass compared to wild type plants since
they grew faster at early stage although they were phe-
notypically normal before bolting. Plants from three T3
transgenic lines showed highly significant increases in
both fresh weights and biomass compared with the cor-
responding control lines (Figure 6). These increases
were all statistically significant at 99.9% confidence
(α-value of 0.001). Average of fresh weight from three
transgenic lines was 507 mg/plant at 4-weeks of age,
corresponding to 125% of the control lines, and average
of biomass of these transgenic lines was 62 mg/plant,
which is also 125% of controls. These data suggested
that overexpression of PtrSUS1 in transgenic Arabidop-
sis plants resulted in increase in biomass perhaps due to
increased SUS activity.
3.5. Effects of PtrSUS1 Overexpression on
Seedling Growth under Various Sucrose
To investigate whether PtrSUS1-overexpressing plants
have elevated sucrose tolerance during seedling devel-
opment, the same three transgenic lines as described
above were selected for comparison in SUS enzyme ac-
tivity and growth with the wild type control in response
to varying concentrations of sucrose. Seeds were germi-
nated on half-strength MS media containing different
levels of sucrose in both normal light and dark condi-
Under normal light and in the media containing 1-2%
sucrose, although visible increases of root growth from
transgenic plants were observed, no highly statistical
significances were found between transgenic lines and
wild type (Figure 7(a)). However, with the increased
sucrose concentrations, especially greater than 3%, the
root growth under normal light was significantly higher
in transgenic than wild type plants (Figure 7(a)). It is
interesting that root growth under dark condition showed
statistically significant differences at all sucrose concen-
trations between transgenic and wild type plants (Figure
7(b)). Hypocotyls in dark showed slight or significant
increase for different transgenic lines and treatments
with the trend of more statistically significance found in
higher sucrose concentrations (Figure 7(c)). All trans-
genic and wild type plants exhibited extremely slow
growth in media without sucrose, indicating the impor-
tance of sucrose for the growth of young seedlings, and
only growth of roots in the dark showed statistically sig-
nificant differences between transgenic and control
plants (Figure 7(b)). They all showed growth inhibition
in media with 4-8% sucrose with the most severely re-
duced growth for wild type plants either in normal light
or dark conditions (Figure 7). Roots of wild type were
much shorter than those of three transgenic lines, and so
were hypocotyls in dark (hypocotyls were too short to be
measured accurately in light condition). These results
indicate that overexpression of PtrSUS1 results in en-
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
Figure 6. Fresh weight (a) and biomass of whole plant (b) for aspen SUS transgenic Arabidopsis
plants grown in greenhouse. Mean ± SE was calculated from 4-week-old 10 plants per transgenic
line and wide-type (Col-0). Three asterisks ***indicate highly significant difference at the 99.9%
hanced seedling tolerance to higher sucrose concentra-
tions in the growth medium.
3.6. Effect of Constitutive PtrSUS1 Expression
on SUS Activity, Fresh Weight and Biomass
in Response to Different Osmotic Stresses
To further determine the effect of PtrSUS1 transgene
expression on SUS activities and correlated changes on
biomass in response to different levels of sucrose, prog-
eny of three transgenic lines and wild type plants were
germinated and cultivated in vertical Petri dishes as de-
scribed above under increasing serial concentrations of
sucrose. As we have seen above, all transgenic and wild
type Arabidopsis plants showed normal growth in media
with 1-3% sucrose and displayed reduced root and hy-
pocotyl length, and a general growth inhibition in re-
sponse to higher sucrose concentrations from 4-8%, and
were unable to grow in the concentration of 10% sucrose
(data not shown). These inhibitory effects increased pro-
gressively with increasing sucrose concentration, and the
wild type plants reduced growth much more rapidly than
transgenic plants (Figure 7). SUS enzyme activities were
assayed for protein extracts of 15-d-old three transgenic
and wild type plants in response to three sucrose con-
centrations of 1, 4, and 8%, and the results are shown in
Figure 8. Overall, SUS activity increased with the eleva-
tion of sucrose concentration in growth media. Trans-
genic plants of T3-6 and T3-11 showed slight increase of
SUS activity when growing in 1% of sucrose, and only
T3-7 showed significant difference compared with wild
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
Figure 7. Root length in light condition (a), root length in dark condition (b) and hypocotyls
height in dark condition (c) in transgenic Arabidopsis plants carrying and expressing aspen
SUS gene in various sucrose concentrations. Mean ± SE was calculated from 10 plants per
transgenic line and wide-type (Col-0). One asterisk *indicates significant difference at the
95% level; two asterisks **indicate very significant difference at the 99% level; three aster-
isks ***indicate highly significant difference at the 99.9% level.
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
Figure 8. Comparison of SUS activity between wild-type and three PtrSUS1 transgenic lines of
Arabidopsis thaliana under different sucrose concentrations. Effect of sucrose concentrations
and PtrSUS1 transgene on SUS activity, measured from the 15-d-old whole plants grown in
0.5x MS medium in vertical Petri dish plates. SUS activity was higher in transgenic plants and
treatments with higher concentrations of sucrose. Error bars indicate the standard error (SE: n =
3). Two asterisks **indicate very significant difference at the 99% level; three asterisks ***in-
dicate highly significant difference at the 99.9% level.
type. However, T3-6 and T3-11 showed significant in-
crease at 99%, and T3-7 at 99.9% confidence in response
to 4 and 8% sucrose. Thus SUS activity of PtrSUS1
transgenic lines increased to a larger degree in response
to higher levels of sucrose compared to that of wild type.
The above plants were also used for measuring root
length in normal light in different levels of sucrose were
harvested for further measuring fresh weight and bio-
mass (dry weight) when they were 3-week-old. As
shown in Figure 9, higher sucrose concentrations sig-
nificantly decreased the fresh weight and dry biomass of
wild type plants than at 3% sucrose, but relatively, did
not dramatically affect those of the transgenic plants.
Both the fresh weight and the biomass of the transgenic
plants were higher than those of the wild type plants in
all concentrations of sucrose. Similarly as we have seen
before, differences between transgenic and wild type
plants became more drastic with the increase of sucrose
especially when reaching 4%. Statistically significant
differences in fresh weight were observed for all sucrose
treatments between transgenic lines and wild type plants,
and so was the significant difference in biomass for most
of sucrose levels except 2%.
In this study, we wanted to examine the effect of the
PtrSUS1 overexpression on growth and development of
transgenic Arabidopsis plants. PtrSUS1 was successfully
transformed into Arabidopsis plants as confirmed by
genomic PCR and PtrSUS1 overexpression confirmed
through RT-PCR using PtrSUS1 specific primers. Three
selected transgenic lines (T3-6, T3-7 and T3-11) showed
33%, 39%, and 43% increase, respectively, in SUS ac-
tivity at the whole plant level. These results are consis-
tent with the increase in total SUS transcript levels due to
the contribution of PtSUS1 (Figure 3). This increase was
sufficient to affect and change the sucrose content and
metabolism of the Arabidopsis plants and thus affect the
growth and development, as Coleman et al. [24] pointed
out that a small increase in the expression of SUS gene
caused a significant effect on carbohydrate levels.
No significantly different visible phenotypes were
observed in transgenic PtrSUS1 plants compared to wild
type especially after the growth for four weeks. However,
the faster growth and about 40% increased biomass in
transgenic plants suggested that the overexpression of
PtrSUS1 gene had a significant impact on plant growth
in Arabidopsis. All three transgenic lines showed sig-
nificant increases in root length (dark and light condi-
tions), hypocotyl length (dark), fresh weight and bio-
mass compared to the wild type plants. An increase in
plant height was also reported with the over-expression
of SUS in poplar under the control of the cauliflower
mosaic virus 35S promoter [3] and in tobacco either un-
der the single 35S, double 35S promoter or 4CL pro-
moter [24]. It is reasonably believed that the overexpres-
sion of SUS followed by the increased SUS enzyme ac-
tivity enhances the capacity of plants to more effectively
use photosynthates and produce more biomass. SUS has
been identified as an indicator of sink strength [30], so it
is possible that the greater increase of sink strength
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
Figure 9. Fresh weight (a) and biomass of whole plant in light condition (b) for aspen SUS
transgenic Arabidopsis plants in various sucrose concentrations. Mean ± SE was calculated
from 10 plants per transgenic line and wide-type (Col-0). One asterisk *indicates significant
difference at the 95% level; two asterisks **indicate very significant difference at the 99%
level; three asterisks ***indicate highly significant difference at the 99.9% level.
caused by the overexpression of SUS promotes plant
growth. This suggests the possibility of increasing con-
vertible biomass for biofuel resources through manipu-
lating SUS gene.
There exist some controversy about the roles of indi-
vidual SUS in the biosynthesis of starch and cellulose,
and plant growth and development. In the suppression
studies of SUS gene expression and enzyme activity, a
decrease in starch and/or cellulose content has been
demonstrated in potato [1,17], in maize [38], in carrot
[39,40], in pea [41], and in cotton [31]. In contrast, the
lack of phenotypic changes in the single and multiple
knockout lines of AtSUS1, AtSUS2, AtSUS3, and AtSUS4
isoforms in Arabidopsis [42] has brought uncertainty
whether this enzyme is necessary in starch and cellulose
biosynthesis. This could be due to plasticity in plant me-
tabolism and utilization of alternative pathways of su-
crose cleavage such as invertase, the transcript of which
is very high in Arabidopsis sink tissues [43]. Reverse
genetics approaches, hence, should be further used to
provide more and definitive evidence for the importance
of SUS in Arabidopsis. On the other hand, the results
from the studies of SUS upregulation are not consistent.
The over-expression of cotton SUS in tobacco showed an
F. Y. Xu et al. / Advances in Bioscience and Biotechnology 1 (2010) 426-438
Copyright © 2010 SciRes. ABB
increase in starch accumulation in transgenic lines with
the promoter of 4CL but no significant increase was ob-
served in other lines with 35S promoter [24]. In the same
experiment, only one transgenic line with the double 35S
promoter showed a significant increase in cellulose con-
In this study, we also observed that PtrSUS1 trans-
genic Arabidopsis plants have the feature of early bolt-
ing and flowering compared with wild type. The role of
SUS in reproductive development has been verified from
several studies. SUS activity was severely inhibited by
the antisense RNA in flowers and fruit pericarp tissues in
antisense transgenic tomato plants, and in turn causing a
reduced fruit set and a slower growth rate of fruits [30].
These results suggest that SUS participates in regulating
sucrose import capacity of young tomato fruit, which is a
determinant for fruit set and development. RNAi ap-
proach was successfully used to silence the expression of
SUS gene in the endosperm and led to the arrest of early
seed development in cotton [44]. While studying the
differential expression of three SUS genes during fruit
development in citrus, Komatsu et al. [45] reported that
SUS activity in edible tissue was high in the early stages
and decreased until mid-development, then rapidly in-
creased during maturation. The increase in activity dur-
ing maturation paralleled that of sucrose accumulation,
indicating the important role of SUS on sugar metabo-
lism when sucrose is accumulated in fruit. Based on en-
zyme activity, transcript levels, and protein localization,
the importance of SUS for sucrose metabolism has been
demonstrated in starch-storing reproductive structures of
legumes such as Vicia [46] and Pisum sp. [41], in
developing nectaries of ornamental tobacco [47], and in
cotton seeds [19,48]. High SUS activity was also found
in reproductive sink tissues of several other plant species
including Arabidopsis silique [5], canola seed and
silique [49], cotton fiber and seed [49,50], wheat grain
[51], rice [52], and tomato fruit [18]. High SUS activity
in these reproductive tissues suggests an important role
of SUS enzyme for sucrose metabolism in determining
sink strength. Fallahi et al. [5] pointed out that during
early stages of seed development, high SUS activity is
needed to establish a strong sink for sucrose by break-
down of imported sucrose to fructose and UDP-glucose.
The observed increased tolerance to higher sucrose
stress of the PtrSUS1 transgenic plants were positively
correlated with the increased SUS activities. We showed
here that, under high sucrose stress, transgenic plants
over-expressing PtrSUS1 cleaved more sucrose, as de-
duced from their higher root and hypocotyls growth, and
higher fresh weight and biomass per plant in comparison
with the wild type plants (Figure 5, 7, and 9). This in-
creased tolerance to higher sucrose or lower osmotic
pressure is closely related to the over-expression of
PtrSUS1 and correspondingly increased SUS activities
in these transgenic Arabidopsis lines, suggesting the role
of SUS in cleaving sucrose. This increase is further
strengthened because the SUS expression was upregu-
lated by osmotic stress rather than by the increase in
sucrose concentration per se [53,54]. Moreover, recent
studies also suggest that AtSUS1 gene is regulated by
sugars (sucrose, glucose, and mannose) through hexo-
kinase-dependent mechanisms [55]. Sugars are known to
be inducers/repressors of gene expression and they have
key roles in signal transduction pathways [56,57]. As a
conclusion, the regulation of AtSUS1 possibly involves
two or more transduction pathways depending on the
nature and strength of the signal [55].
In summary, the current work provided new informa-
tion to advance our overall knowledge about the effect of
heterologous SUS on transgenic Arabidopsis plants and
their responses to various concentrations of sucrose. We
demonstrated here that the over-expression of an aspen
SUS (PtrSUS1) alone in Arabidopsis enhanced SUS ac-
tivity, indicating that the foreign SUS gene functions in a
similar manner to the endogenous AtSUS activity. The
increased SUS activity in turn changed sucrose metabo-
lism inside the transgenic plants. The observed pheno-
types with faster growth and early flowering as well as
tolerance-enhanced in high sucrose are closely correlated
with the increased cleavage capacity of SUS for sucrose
in plants. The present results emphasize the importance
of SUS-mediated sucrose cleavage process in response
to high sucrose concentrations, and provide a basis to
better understanding of the integrated functions of SUS
during plant growth and development.
This work was supported by a grant from National Science Founda-
tion’s CAREER program (IBN- 0236492). This work was also partially
supported by the World Class University project of the Ministry of
Science and Technology of Korea (R31-2009-000-20025-0). We wish
to thank Sandra Hubscher and Rama Joshi for critical reading of this
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