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Journal of Plant Research ISSN 0918-9440Volume 126Number 5 J Plant Res (2013) 126:743-752DOI 10.1007/s10265-013-0562-6
Altered invertase activities of symptomatictissues on Beet severe curly top virus(BSCTV) infected Arabidopsis thaliana
Jungan Park, Soyeon Kim, EunseokChoi, Chung-Kyun Auh, Jong-BumPark, Dong-Giun Kim, Young-JaeChung, Taek-Kyun Lee & Sukchan Lee
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REGULAR PAPER
Altered invertase activities of symptomatic tissues on Beet severecurly top virus (BSCTV) infected Arabidopsis thaliana
Jungan Park • Soyeon Kim • Eunseok Choi •
Chung-Kyun Auh • Jong-Bum Park • Dong-Giun Kim •
Young-Jae Chung • Taek-Kyun Lee • Sukchan Lee
Received: 11 October 2012 / Accepted: 18 March 2013 / Published online: 16 April 2013
� The Botanical Society of Japan and Springer Japan 2013
Abstract Arabidopsis thaliana infected with Beet severe
curly top virus (BSCTV) exhibits systemic symptoms such
as stunting of plant growth, callus induction on shoot tips,
and curling of leaves and shoot tips. The regulation of
sucrose metabolism is essential for obtaining the energy
required for viral replication and the development of
symptoms in BSCTV-infected A. thaliana. We evaluated
the changed transcript level and enzyme activity of inver-
tases in the inflorescence stems of BSCTV-infected A. tha-
liana. These results were consistent with the increased
pattern of ribulose-1,5-bisphosphate carboxylase/oxygen-
ase activity and photosynthetic pigment concentration in
virus-infected plants to supply more energy for BSCTV
multiplication. The altered gene expression of invertases
during symptom development was functionally correlated
with the differential expression patterns of D-type cyclins,
E2F isoforms, and invertase-related genes. Taken together,
our results indicate that sucrose sensing by BSCTV infec-
tion may regulate the expression of sucrose metabolism and
result in the subsequent development of viral symptoms in
relation with activation of cell cycle regulation.
Keywords A. thaliana � Beet severe curly top virus �D-type cyclin � E2F � Invertase
Introduction
Beet severe curly top virus (BSCTV) has a monopartite
genome of single-stranded DNA and is a member of
Curtovirus of the geminivirus family. The BSCTV viral
genome code seven open reading frames (ORFs) whose
protein products are involved in viral structure and insect
vector transmission: capsid protein (V1); replication (rep
C1 and C3); movement (V1 and V3); ssDNA accumulation
(V2); and symptom development (C2 and C4) (Briddon
et al. 1989; Frischmuth et al. 1993; Hormuzdi and Bisaro
1993, 1995; Stanley et al. 1992). Typical viral symptoms
include leaf curling, vein yellowing, and growth stunting in
various host plants. BSCTV causes systemic symptoms in
Arabidopsis thaliana, including shoot curling, swelling bolt
and growth stunting, and more severe symptoms have been
reported such as anthocyanin accumulation and callus-like
structure formation in symptomatic tissues (Lee et al.
1994). Viruses can be a key modulator of plant develop-
ment during symptom development. The pathomorpho-
genesis is a very complicated process in whole plant
J. Park and S. Kim contributed equally to this work.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10265-013-0562-6) contains supplementarymaterial, which is available to authorized users.
J. Park � S. Kim � E. Choi � S. Lee (&)
Department of Genetic Engineering, Sungkyunkwan University,
Suwon 440-746, Korea
e-mail: [email protected]
C.-K. Auh
Division of Life Sciences, Mokpo National University,
Muan 534-729, Korea
J.-B. Park � D.-G. Kim
Department of Biological Science, Silla University,
Busan 617-736, Korea
Y.-J. Chung
Department of Life Science and Biotechnology,
Shin Gyeong University, Hwaseong 445-741, Korea
T.-K. Lee (&)
South Sea Environment Research Department, Korea Ocean
Research and Development Institute, Geoje 656-830, Korea
e-mail: [email protected]
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DOI 10.1007/s10265-013-0562-6
Author's personal copy
development, but little is currently known regarding the
development of these symptoms (Lee et al. 1994).
Most cells in developing plants have left the cell division
cycle and are not in the proper state to support viral DNA
replication. The ability of a virus to alter the cell cycle
would greatly expand the cellular range of viral infection.
Some of the geminiviruses, including Beet curly top virus
(BCTV), Abutilon mosaic virus, and Squash leaf curl virus
are phloem-limited and may replicate in procambial cells
competent for DNA replication, but it is clear that other
geminiviruses such as Tomato golden mosaic virus
(TGMV) are not limited to the phloem, and can replicate in
differentiated tissues throughout the leaf, stem, and roots of
plants (Hanley-Bowdoin et al. 2000; Stanley 1991). The
virus is presumably capable of activating the host replica-
tion machinery, and this presumption is supported by data
showing the accumulation of proliferating nuclear antigen
(PCNA), the processivity factor of host DNA polymerase,
in differentiated cells of TGMV infected-plants (Nagar
et al. 1995). Ascencio-Ibanez et al. (2008) demonstrated
that geminiviruses modulate plant cell cycle status by dif-
ferentially impacting the D-type cyclin (CYCD), retino-
blastoma-related protein, and E2F regulatory network and
facilitate progression into the endocycle in the Cabbage leaf
curl virus (CaLCuV) system on A. thaliana.
In addition, BSCTV-infected A. thaliana requires more
energy to produce actively dividing cells and callus-like
structures on the shoot tips and axillary buds (Park et al.
2004, 2010). Hence, the regulation of sucrose metabolism
in BSCTV-infected A. thaliana is essential as an energy
source for viral replication and symptom development. The
key sucrose metabolism enzymes are sucrose synthase, and
invertase. Sucrose synthase (UDP-glucose: D-fructose
2-glucosyl-transferase EC.2.4.1.13) is a glycosyl transfer-
ase that converts sucrose into UDP-glucose and fructose
via reversible hydrolysis (Tymowska-Lalanne and Kreis
1998). Invertase (EC.3.2.1.26), which is also referred to as
b-fructofuranosidase, breaks sucrose into glucose and
fructose via irreversible hydrolysis. Plant invertases are a
group of multiple isoforms that show characteristic solu-
bility, subcellular localization, and optimum pH. Three
representative types of invertases include a soluble cyto-
plasmic neutral invertase, a soluble vacuolar acid invertase,
and an insoluble cell wall acid invertase (Ruan et al.
2010; Tymowska-Lalanne and Kreis 1998). They are
expressed in development-specific or organ-specific man-
ners. Expression of the invertase genes is modulated by a
variety of factors, including sugars, pathogen infection,
wounding, gravity, and low temperature (Ruan et al. 2010;
Tymowska-Lalanne and Kreis 1998). Sturm and Chrispeels
(1990) reported that the mRNA level of cell wall invertase
increases rapidly in the leaves and roots of carrot infected
with Erwinia carotovora. The infection of the roots and
leaves of carrot plants with E. carotovora result in a rapid
increase in mRNA levels with maximum expression after
1 h. Benhamou et al. (1991) demonstrated that invertase
accumulation was noted in 48 h in the resistant tomato root
after inoculation with fungal wilt pathogen and appeared to
achieve maximum levels 72 h after inoculation. SNF1-
related protein kinase-1 (SnRK1) performs a critical
function in the global control of sucrose metabolism
(Hao et al. 2003). SNF1 phosphorylates and inactivates key
enzymes that control sucrose synthesis (Sugden et al.
1999). Hao et al. (2003) reported that AL2 and L2 gem-
iniviruses interact with and inactivate SNF1 kinase.
We previously analyzed the relative gene expression of
sucrose metabolism-related genes and cell cycle-related
genes in BSCTV C4-overexpressing transgenic plants
(Park et al. 2010). In this BSCTV C4 transgenic A. thali-
ana, the pseudosymptoms induced by the C4 protein
are quite similar to the symptoms of BSCTV-infected
A. thaliana, including the formation of callus-like struc-
tures, C4-induced abnormal cell division, and altered cell
fate in a variety of tissues depending on the C4 expression
level. BSCTV-induced calli were apparently formed as the
result of an uncontrolled cell cycle. The production of more
energy in the host plant is required for de novo cell division
in non-meristematic or fully differentiated tissues after
viral infection. BSCTV infection appears to be associated
with the cell cycle of host plants mediated by changing cell
cycle marker gene expression and genes involved in
sucrose metabolism. Therefore, we analyzed the expression
patterns of cell cycle-related genes and sucrose metabolism
genes in this study.
Materials and methods
Plant growth and virus infection
A. thaliana seeds were planted in flats containing soil,
covered with plastic domes, and stratified for 3 days at
4 �C. The plants were transferred to growth chambers
operating at 23–25 �C, 60–80 % relative humidity, with a
day length of 10–12 h supplied by fluorescent bulbs at an
intensity of 100–200 lE m-2 s-1. The covers were
removed 1–2 weeks after planting, and the seedlings were
watered and fertilized as needed. Four to five week old
plants were inoculated with infectious viral DNA by
agroinfection of wounds produced on the crown of the
rosette by needle puncture as described previously (Lee
et al. 1994). Agrobacterium strain GV3101 (Mock) and the
pBSCTV clone (pMON vector) containing the BSCTV
genome dimer in Agrobacterium were inoculated on the
rosette centers of 4–5-week old A. thaliana ecotype Col-0
via agroinfection (Grimsley et al. 1987; Grimsley 1990).
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Inflorescence stems were harvested 7, 14, and 21 days
post-inoculation.
Quantitative real-time RT-PCR
Each of the plant tissues was harvested 7, 14, and 21 days
post-inoculation and frozen in liquid nitrogen. Total RNA
was isolated from leaves harvested from 10 plants using a
QIAGEN RNeasy Plant Mini Kit (Qiagen, Valencia, CA,
USA). The cDNA strand was synthesized from 5 lg of
total RNA using oligo (dT) primers and Moloney murine
leukemia virus reverse transcriptase (Promega, Madison,
WI, USA) for the quantitative real-time RT-PCR and semi-
quantitative RT-PCR. The actin 2 and 18S rRNA mixture
was diluted 10 times with water to reduce the high-abun-
dance effects on PCR amplification. Reverse transcription
was conducted with 5 lg of RNA, and primers from
Supplementary Table 1, designed by Primer3 (http://www-
genome.wi.mit.edu/cgibin/primer/primer3.cgi/primer3_
www.cgi) and synthesized by Bioneer (Bioneer Co., Seoul,
Korea). Each data point represents the average of three or
four experiments and error bars indicate standard devia-
tions, unless stated otherwise. The gene expression patterns
between mock and infected groups determined by quanti-
tative real-time RT-PCR using a Rotor Gene 3000 (Corbett
Research Co., Sydney, Australia) program and semi-
quantitative RT-PCR were distinguished using the patterns
detected by 1.5 % agarose gel electrophoresis.
Rubisco assay
Frozen plant samples were ground in 10 volumes of
grinding buffer [100 mM bicine, pH 8.0, 25 mM MgCl2,
0.01 mM leupeptin, 1 mM PMSF, 1 mM Na-EDTA, 0.5 %
(v/v) b-mercaptoethanol, and 12.5 % glycerol]. Rubisco
activity was assayed in a mixture containing 50 mM Tris–
HCl (pH 8.0), 30 mM MgCl2, 5 mM DTT, 0.2 mM
NaH14CO3, supernatant enzyme extracts, and 5 mM RuBP.
The reaction was incubated for 5 min at 25 �C, and halted
by the adding 5 N NaOH. The reaction mixture was dried,
resuspended with ddH2O, and added to a scintillation
cocktail and counted (Ou-Lee and Setter 1985).
Invertase and sucrose synthase activity assay
Frozen plant tissues were ground in liquid nitrogen, and
homogenization buffer 1 (200 mM HEPES, 3 mM MgCl2,
1 mM EDTA and 2 % glycerol) was added to 1 g of
ground samples, and the crude extracts (CEX) were
extracted. After washing the pellets, homogenization buffer
2 (200 mM HEPES, 3 mM MgCl2, 15 mM EDTA, 2 %
glycerol, 1 M NaCl, 0.1 mM PMSF and 0.01 mM ben-
zamidine) was added, and the cell wall extracts (CWEX)
were extracted after an overnight incubation at 4 �C. CEX
and CWEX were reacted in 0.1 M sucrose KPO4 citrate
buffer at pH values of 4.5 and 6.8, respectively, at 4 and
25 �C (Roitsch et al. 1995). Glucose concentration was
measured using Sigma diagnostics glucose reagents for
quantitative and enzymatic determinations at 520 nm
(Sigma, procedure No. 115). Sucrose synthase activity was
determined as the UDP-Glc-dependent sucrose formation
in the presence of fructose. SPNT enzyme precipitates were
incubated with 7.5 mM UDP-Glc, 7.5 mM fructose, and
15 mM MgCl2 in 50 mM HEPES-NaOH buffer (pH 7.5,
25 �C), then incubated with invertase (pH 4.5, 25 �C), and
the concentration of glucose was measured as described for
the invertase method (Ou-Lee and Setter 1985).
Pigment determination
The extraction and the determination of chlorophyll a and
carotenoid concentrations were conducted via spectropho-
tometric analysis (Agilent 8453 System, Hewlett-Packard
Co. Palo Alto, CA, USA). The plant samples were ground
in liquid nitrogen and extracted with 90 % acetone via
centrifugation. The absorbance of the decanted superna-
tants was measured at the following wavelengths without
delay: 750, 664, 647, 630, 510, and 480 nm, and the
amount of pigments were calculated (Parsons et al. 1984).
Statistical analysis
Statistical analyses were performed using GraphPad Prism
5 software package (GraphPad Software, USA) and
included a one-way ANOVA with a Tukey’s Multiple
Comparison Test for significant differences.
In silico analysis of the BSCTV intergenic region (IR)
The promoter sequences were analyzed for the presence of
plant cis-regulatory elements using the plantCARE data-
base, as described previously (Lescot et al. 2002).
Results
Callus-like structures induced in BSCTV-infected
A. thaliana
BSCTV symptoms appeared on A. thaliana 1 week after
infection on newly developing inflorescence stems, and the
symptoms developed for an additional 2 weeks. Figure 1
shows that the BSCTV-infected A. thaliana had severe
stunting of stems and leaves (Fig. 1b), and callus-like
structures on the stems (Fig. 1c, d). These two disease
symptoms were very unique in the BSCTV-infected
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A. thaliana samples, but no such results have been reported
on other host plants. The lifespan of A. thaliana ecotype
Col-0 is approximately 6–8 weeks. Thus, if 4–5-week old
A. thaliana samples were infected with BSCTV, the dis-
eased plants shown in Fig. 1b were normally at the end of
their life cycle. Thus, mock-inoculated 8-week old A. tha-
liana samples usually turned brown with siliques on stems,
and the rosette leaves were almost in the last stage of
senescence.
However, the BSCTV-infected plants showed a change
in rosette leaf color from green to brown or yellow 7 days
post-inoculation and newly developing leaves were green-
ish or dark green. De novo cell division and callus forma-
tion on symptomatic tissues were initiated 14–20 days post-
inoculation. Well-developed callus structures were mainly
observed in the petioles and shoot tips 21 days post-inoc-
ulation (Fig. 1c, d). At this stage, BSCTV-infected A. tha-
liana continued to produce more new rosette leaves and
maintained more new greenish rosette leaves than those in
mock-inoculated plants.
Expression of cell cycle-related genes
BSCTV-infected plants showed de novo cell division,
mostly on the shoot tips and axillary buds (Fig. 1). To
understand the relationship between cell division and
D-type cyclin expression induced by BSCTV, quantita-
tive real-time reverse transcription polymerase chain
reaction (RT-PCR) was carried out with 4 D-type cyclin-
specific primers and RNA extracted from the shoot tips
at 3 weeks after BSCTV inoculation. BSCTV-infected
plants induced gene expression of 1 D-type cyclin
(cycD3;1) but cycD2;1 decrease (Fig. 2a). cycD1;1 and
cycD4;1 gene expression in BSCTV-infected plants was
not changed relative to that in the mock-inoculated
plants.
Figure 2b shows that the E2F genes were differentially
expressed according to the BSCTV infection. E2Fb was
induced four times more in BSCTV-infected plants relative
to that in mock-inoculated plants. E2Fc decreased in
BSCTV-infected plants relative to that in mock-inoculated
plants. However, the E2Fa transcripts accumulated three
times more in BSCTV-infected plants than those in the
mock-inoculated plants. If BSCTV infects A. thaliana, the
C1 protein binds to Rb and releases E2F, which activates
cell cycling. E2F and cyclin overexpression of cell cycle
modulators lead to the prompt cycling of the mitotic cell
cycle. In silico analysis of the BSCTV intergenic region
(IR) showed that one of the E2F binding motifs was present
(Fig. 3; Supplementary Table 2).
Expression of invertase-related genes
Quantitative real-time RT-PCR results demonstrated that
the expression of ivr1 among four invertase isomers (ivr1,
ivr3, ivr4, and ivr6) increased four times in the BSCTV-
infected shoot tips of A. thaliana 21 days post-inoculation
(Fig. 4). The other two invertase isomers (ivr3 and ivr4)
did not changed expression patterns, regardless of whether
the plants were BSCTV-infected or mock-inoculated. ivr6
expression decreased in BSCTV-infected plants (Fig. 4).
The expression patterns of five invertase isomers differed
based on their optimal pH, temporal, and/or spatial
expression, and BSCTV infection.
Fig. 1 Symptoms caused by BSCTV infection in A. thaliana.
Agrobacterium strain GV3101 containing a dimer of the BSCTV
genome, pBSCTV, was inoculated in 4–5-week old A. thaliana
ecotype Col-0 via agroinfection. a Mock-inoculated A. thaliana
showed normal growth at 14 days post-inoculation and produced
shoots. b BSCTV-infected A. thaliana showed symptoms of curling
stems and cauline leaves, and anthocyanin accumulation on axillary
buds 2–3 weeks post inoculation. c, d BSCTV-infected A. thaliana
had swollen stems due to hyperplasia and callus-like structures on
shoot tips
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Altered rubisco activity and photosynthetic pigment
concentration
Inflorescence stems were harvested at 3 weeks after
BSCTV infection and analyzed to measure rubisco activity.
Rubisco activity of inflorescence stems changed slightly in
the BSCTV-infected plants compared to that in mock-
inoculated plants (Fig. 5). It has been previously reported
that the photosynthetic rate and rubisco activity declines by
20–50 % in BCTV-infected sugar beet and Nicotiana
benthamiana (Swiech et al. 2001). However, in our exper-
iment, a 15 % increased pattern of rubisco activity 21 days
post-inoculation A. thaliana was related with specific
development stages of BSCTV-infected A. thaliana.
The concentration of photosynthetic pigments was
measured in the inflorescence stems of BSCTV-infected A.
thaliana (Fig. 5). Fully mature BSCTV infected rosette
leaves showed symptoms of yellowing. Additionally, the
newly developing small leaves curled but remained green.
The inflorescence stems accumulated 40–50 % more
chlorophyll than that in mock-inoculated A. thaliana 7 and
14 days post- BSCTV inoculation. The chlorophyll a and
carotenoid accumulation patterns were similar to those
observed during the post-inoculation stage.
BSCTV infection maintained invertase and sucrose
synthase activity
The activities of invertase and sucrose synthase were
measured 7, 14, and 21 days post-inoculation to evaluate
the sucrose metabolism activity after BSCTV infection.
The activity of the two types of invertases such as cell wall
invertase and neutral invertase was measured separately
(Fig. 6).
Fig. 2 Gene expression of
D-type cyclins and E2Fs.
D-type cyclin and E2F genes
were regulated by BSCTV-
infected A. thaliana,
respectively. a Four D-type
cyclins (cycD1;1, cycD2;1,
cycD3;1, and cycD4;1) and
b three E2Fs (E2Fa, E2Fb, and
E2Fc). Asterisks indicate
statistical significance
determined by Student’s t test
(p B 0.05)
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Cell wall and neutral invertase activities were sub-
stantially higher in the BSCTV-infected A. thaliana than
those in mock-inoculated samples (Fig. 6). Inflorescence
stems that produced more severe symptoms, such as de
novo cell division and callus-like structures, did not show
reduced activity. Sucrose synthase (SuSy) activity in
BSCTV-infected plants increased slightly over that in
mock-inoculated plants, but no dramatic change was
observed, and a similar level of SuSy activity was
maintained (Fig. 6).
Discussion
In our A. thaliana and BSCTV experimental system, ivr1
showed high transcript expression levels in the shoot tips of
BSCTV-infected A. thaliana relative to other invertase
isoforms (Fig. 4a). Huang et al. (2007) reported that Ivr1 is
regulated tightly at both the transcriptional and post-tran-
scriptional levels. The total activity of invertase at the post-
transcriptional level is regulated by compartment-specific
inhibitor proteins in a pH-dependent manner (Hothorn et al.
Fig. 3 In silico analysis of the
E2F motif in the BSCTV
intergenic region (IR). The
BSCTV IR sequences were
analyzed for the presence of
plant cis-regulatory elements
using the PlantCARE database
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2010). Considering the respective roles of each invertase
during plant growth and development, the results noted in
the BSCTV-infected A. thaliana samples 21 days post-
inoculation showed altered invertase expression as a result
of viral infection.
Invertases, either alone or in combination with plant
hormones, regulate many aspects of plant growth and
development from gene expression to long-distance nutri-
ent allocation. Invertases are involved in the regulation of
carbohydrate partitioning, developmental processes, hor-
mone responses, and biotic and abiotic interactions (Godt
and Roitsch 1997; Goetz et al. 2000; Lana et al. 2004;
Roitsch and Gonzalez 2004). A continually growing
number of studies have demonstrated that invertases
Fig. 4 The altered expression patterns of invertase-related genes.
Invertase isoforms (ivr1, ivr3, ivr4, and ivr6) gene expression was
analyzed via quantitative real-time RT-PCR in the shoot tips of
BSCTV-infected plants and mock-inoculated plants. Total RNAs
from BSCTV-infected and mock-inoculated plants were harvested
3 weeks after inoculation, 5–6 weeks after germination. The quanti-
tative comparison was accomplished by adjusting RNA concentra-
tions using the 18S rRNA and actin 2 genes as an internal control.
Asterisks indicate statistical significance determined by Student’s
t test (p B 0.05) Fig. 5 Rubisco activity and pigment deposition. Rubisco enzyme
activity was measured from the inflorescence stems of BSCTV-infected
A. thaliana 7, 14, and 21 days post-inoculation. The deposition and
accumulation (or degradation) of chlorophyll a and carotenoid photo-
synthetic pigments were quantified in inflorescence stems of BSCTV-
infected A. thaliana 7, 14, and 21 days post-inoculation. Chlorophyll a
and carotenoids were extracted from the tissues with different organic
solutions. Results are expressed as means from three independent
experiments. Results expressed as mean ± standard deviation obtained
from three independent experiments. Asterisks indicate statistical
significance determined by Student’s t test (p B 0.05)
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perform critical functions in various aspects of plant
growth and development (Gonzalez et al. 2005; Heyer et al.
2004; Wang et al. 2008), but little information is available
about pathomorphogenesis, which is also important during
the plant life cycle. Therefore, our observations on the
BSCTV A. thaliana system, specifically regarding the
regulation of invertase activity and gene expression could
provide a model system for understanding the control of
sucrose metabolism by plant viruses.
The effects of a pathogen infection on modulating
invertase activity have also been addressed in the context
of plant interactions with bacteria or fungi (Benhamou
et al. 1991; Joosten et al. 1991; Sturm and Chrispeels
1990). Sturm and Chrispeels (1990) reported that the
mRNA levels of cell wall invertase increase rapidly in the
leaves and roots of carrot plants infected with the bacte-
rium E. carotovora. Joosten et al. (1991) showed a sys-
temic increase in invertase and a very low concentration of
sucrose in tomato leaves infected with the fungi Clado-
sporium fulvum. The induction of invertase by pathogen
infection may convert infected tissues into sink tissues
when sufficient energy sources are required (Benhamou
et al. 1991).
The relationship between sucrose metabolism and the
defense pathway was suggested by Jang and Sheen
(1994). The model specifies that the activation of
defense-related genes requires the sugar signal transduc-
tion pathway comprised of invertase. It has been dem-
onstrated in fungal systems that the induction of
proteinase inhibitor II and chalcone synthase in the
defense pathway is caused by interactions between car-
bohydrate metabolism and the defense pathway (Herbers
et al. 1996). Moreover, Herbers et al. (2000) reported that
the infection of tobacco plants with potato virus Y
(PVYN) leads to accumulation of soluble carbohydrates,
and sugars act as amplifiers for defense responses during
plant pathogen interactions.
We are able to conclude that BSCTV infection of A.
thaliana results in change in mRNA transcripts and
invertase activity. We were able to determine the gene
expression patterns by analyzing the expected genes based
on viral symptoms without pin-pointing the relationship
and signal transduction between several pathways that
might be involved in the development of BSCTV symp-
toms. In our molecular analysis of cell cycle-related genes,
four D-type cyclin genes showed differential induction
patterns as a result of BSCTV infection (Fig. 2). Cyclins
perform a crucial role regulating cell division by inducing
quick synthesis and are repressed or degraded by other
signals (Dewitte and Murray 2003). However, although
many different cyclins have been isolated from different
plant species and there have been few previous reviews
regarding the functions of cell cycle-related genes during
geminivirus infection (Lee et al. 1994; Park et al. 2004,
2010), we still do not fully understand the in vivo functions
of each cyclin during symptom development observed in
the BSCTV and A. thaliana system.
Fig. 6 Invertase activity in the inflorescence stems of BSCTV-
infected A. thaliana. The sucrose-cleaving activities of cell wall acid
invertase, cytoplasmic neutral invertase, and sucrose synthase were
measured with the BSCTV-infected A. thaliana extracts 7, 14, and
21 days post inoculation. Results are expressed as means from three
independent experiments. Results expressed as mean ± standard
deviation obtained from three independent experiments. Asterisks
indicate statistical significance determined by Student’s t test
(p B 0.05)
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Ascencio-Ibanez et al. (2008) reported that geminivi-
ruses modulate plant cell cycle status by differentially
affecting the CYCD, retinoblastoma-related protein, and
E2F regulatory networks, and facilitating progression into
the endocycle. They demonstrated that cycD4;2 is induced
by CaLCuV infection, but that cycD3;1 and cycD4;1 are
unchanged, whereas CycD1;1 and cycD3;2 are downreg-
ulated. Figure 2 shows that cycD3;1 was induced by 45 %
more after BSCTV infection but that other cycD genes
were suppressed (cycD2;1) or unchanged (cycD1;1 and
cycD4;1). Riou-Khamlichi et al. (2000) proposed that the
control of cycD2 and cycD3 by sucrose functions as a
component of cell cycle control in response to cellular
carbohydrate status.
Interestingly, we found that E2Fa and cycD3;1 were
induced in abnormal callus cells induced by the interaction
between BSCTV infection and A. thaliana (Fig. 2). The
coordinated cycD3;1 and E2Fa expression patterns were
also noted in transgenic A. thaliana with an abnormal
phenotype, which develop by low cycD3;1 and E2Fa
expression level and by the dexamethasone induction sys-
tem (de Jager et al. 2009). Studies have substantiated the
CYCD3-RBR-E2Fa pathway as a key regulator of the G1/S
transition in plants (de Jager et al. 2005; Dewitte et al.
2003). These genes modulated by cycD3;1 induction are
correlate with the role of CYCD3 in promoting S- phage
and its ability to activate the cell cycle (de Jager et al.
2009). Our limited knowledge and information regarding
host gene expression during BSCTV infection in A. thali-
ana led us to assess several gene expression profiles and to
investigate cell cycle and sucrose metabolism-related
genes. These experimental systems will allow us to gain
more insight into the cell cycle regulation controlled by
sucrose contents which are altered by BSCTV infection.
The bioinformatic analysis of BSCTV IR indicated that
the cis-activating motif responsive to the E2F binding
motif was present (Fig. 3). This is the first report regarding
the presence of the E2F binding motif at the (-)168
position in the BSCTV IR. This E2F binding motif in the
BSCTV IR positively supports induction of the cell cycle
followed by the cyclin D-RBR-E2F pathway in the
BSCTV-A. thaliana system. We identified the presence of
the E2F binding motif in BSCTV IR and the induction of
E2F genes, but we still must establish a new experimental
system to more accurately characterize the functions of the
E2F binding motif in BSCTV IR.
Here, we thought that accumulation of invertase after
viral infection might not be induced for a defense action,
but might affect the supply of the carbon source required
during viral multiplication or symptom development.
BSCTV induces very unique disease symptoms such as
hyperplasia, cell division, and secondary growth in A. tha-
liana (Lee et al. 1994; Park et al. 2004, 2010). It is evident
that BSCTV infection alters sucrose metabolism- and cell
cycle-related gene expression. The changes in the invertase
gene expression patterns and the changes in invertase
activities were correlated with the differential regulation of
the cell cycle-related genes (Figs. 2, 4).
These results indicate that molecular and physiological
responses are associated with the viral symptoms devel-
oped as a result of BSCTV infection in A. thaliana. The
manner in which BSCTV induces altered host metabolism
and the consequent development of viral symptoms remain
unknown. However, in addition to our molecular analysis
regarding symptom development, it might be suggested
that BSCTV can efficiently change gene transcription of
host cells to obtain molecules and energy sources for rep-
lication and movement, and may have a profound effect on
symptom development by regulating the sucrose metabo-
lism of host plants.
Acknowledgments This study was supported by a grant from the
iPET (Korea Institute of Planning and Evaluation for Technology in
Food, Agriculture, Forestry, and Fisheries: No.311058-05-1-HD140),
Ministry for Food, Agriculture, Forestry and Fisheries, Republic of
Korea. This work was also supported by the Korea Institute of Ocean
Science & Technology (Project No. PE98821).
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