1 23

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

1 23

Your article is protected by copyright and all

rights are held exclusively by The Botanical

Society of Japan and Springer Japan. This e-

offprint is for personal use only and shall not

be self-archived in electronic repositories. If

you wish to self-archive your article, please

use the accepted manuscript version for

posting on your own website. You may

further deposit the accepted manuscript

version in any repository, provided it is only

made publicly available 12 months after

official publication or later and provided

acknowledgement is given to the original

source of publication and a link is inserted

to the published article on Springer's

website. The link must be accompanied by

the following text: "The final publication is

available at link.springer.com”.

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: cell4u@skku.edu

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: tklee@kordi.re.kr

123

J Plant Res (2013) 126:743–752

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).

744 J Plant Res (2013) 126:743–752

123

Author's personal copy

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

J Plant Res (2013) 126:743–752 745

123

Author's personal copy

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

746 J Plant Res (2013) 126:743–752

123

Author's personal copy

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)

J Plant Res (2013) 126:743–752 747

123

Author's personal copy

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

748 J Plant Res (2013) 126:743–752

123

Author's personal copy

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)

J Plant Res (2013) 126:743–752 749

123

Author's personal copy

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)

750 J Plant Res (2013) 126:743–752

123

Author's personal copy

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).

References

Ascencio-Ibanez JT, Sozzani R, Lee TJ, Chu TM, Wolfinger RD,

Cella R, Hanley-Bowdoin L (2008) Global analysis of Arabid-

opsis gene expression uncovers a complex array of changes

impacting pathogen response and cell cycle during geminivirus

infection. Plant Physiol 148:436–454

Benhamou N, Grenier J, Chrispeels MJ (1991) Accumulation of

{beta}-Fructosidase in the cell walls of tomato roots following

infection by a fungal wilt pathogen. Plant Physiol 97:739–750

Briddon RW, Watts J, Markham PG, Stanley J (1989) The coat

protein of beet curly top virus is essential for infectivity.

Virology 172:628–633

de Jager SM, Maughan S, Dewitte W, Scofield S, Murray JA (2005)

The developmental context of cell-cycle control in plants. Semin

Cell Dev Biol 16:385–396

de Jager SM, Scofield S, Huntley RP, Robinson AS, den Boer BG,

Murray JA (2009) Dissecting regulatory pathways of G1/S

control in Arabidopsis: common and distinct targets of CYCD3;

1, E2Fa and E2Fc. Plant Mol Biol 71:345–365

Dewitte W, Murray JA (2003) The plant cell cycle. Annu Rev Plant

Biol 54:235–264

Dewitte W, Riou-Khamlichi C, Scofield S, Healy JMS, Jacqmard A,

Kilby NJ, Murray JAH (2003) Altered cell cycle distribution,

hyperplasia, and inhibited differentiation in Arabidopsis caused

by the D-type cyclin Cyc3. Plant Cell 15:79–92

Frischmuth S, Frischmuth T, Latham JR, Stanley J (1993) Transcrip-

tional analysis of the virion-sense genes of the geminivirus beet

curly top virus. Virology 197:312–319

Godt DE, Roitsch T (1997) Regulation and tissue-specific distribution

of mRNAs for three extracellular invertase isoenzymes of

tomato suggests an important function in establishing and

maintaining sink metabolism. Plant Physiol 115:273–282

J Plant Res (2013) 126:743–752 751

123

Author's personal copy

Goetz M, Godt DE, Roitsch T (2000) Tissue-specific induction of the

mRNA for an extracellular invertase isoenzyme of tomato by

brassinosteroids suggests a role for steroid hormones in assim-

ilate partitioning. Plant J 22:515–522

Gonzalez MC, Roitsch T, Cejudo FJ (2005) Circadian and develop-

mental regulation of vacuolar invertase expression in petioles of

sugar beet plants. Planta 222:386–395

Grimsley NH (1990) Agroinfection. Physiol Plant 79:147–153

Grimsley N, Hohn T, Davies JW, Hohn B (1987) Agrobacterium-

mediated delivery of infectious maize streak virus into maize

plants. Nature 325:177–179

Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D

(2000) Geminiviruses: models for plant DNA replication,

transcription, and cell cycle regulation. Crit Rev Biochem Mol

Biol 35:105–140

Hao L, Wang H, Sunter G, Bisaro DM (2003) Geminivirus AL2 and

L2 proteins interact with and inactivate SNF1 kinase. Plant Cell

15:1034–1048

Herbers K, Meuwly P, Frommer WB, Metraux JP, Sonnewald U

(1996) Systemic acquired resistance mediated by the ectopic

expression of invertase: possible hexose sensing in the secretory

pathway. Plant Cell 8:793–803

Herbers K, Takahata Y, Melzer M, Mock HP, Hajirezaei M,

Sonnewald U (2000) Regulation carbohydrate partitioning

during the interaction of potato virus Y with tobacco. Mol Plant

Pathol 1:51–59

Heyer AG, Raap M, Schroeer B, Marty B, Willmitzer L (2004) Cell

wall invertase expression at the apical meristem alters floral,

architectural, and reproductive traits in Arabidopsis thaliana.

Plant J 39:161–169

Hormuzdi SG, Bisaro DM (1993) Genetic analysis of beet curly top

virus: evidence for three virion sense genes involved in

movement and regulation of single- and double-stranded DNA

levels. Virology 193:900–909

Hormuzdi SG, Bisaro DM (1995) Genetic analysis of beet curly top

virus: examination of the roles of L2 and L3 genes in viral

pathogenesis. Virology 206:1044–1054

Hothorn M, Van den Ende W, Lammens W, Rybin V, Scheffzek K

(2010) Structural insights into the pH-controlled targeting of

plant cell-wall invertase by a specific inhibitor protein. Proc Natl

Acad Sci USA 107:17427–17432

Huang LF, Bocock PN, Davis JM, Koch KE (2007) Regulation of

invertase: a ‘suite’ of transcriptional and post-transcriptional

mechanisms. Funct Plant Biol 34:499–507

Jang JC, Sheen J (1994) Sugar sensing in higher plants. Plant Cell

6:1665–1679

Joosten JGH, McCarthy JL, Pusey PN (1991) Dynamic and static

light scattering by aqueous polyacrylamide gels. Macromole-

cules 24:6690–6699

Lana MEB, Garcia MCG, Fatima T, Ehneß R, Lee TK, Proels R,

Tanner W, Roitsch T (2004) Extracellular invertase is an

essential component of cytokinin-mediated delay of senescence.

Plant Cell 16:1276–1287

Lee S, Stenger DC, Bisaro DM, Davies KR (1994) Identification of

loci in Arabidopsis that confer resistance to geminivirus

infection. Plant J 6:525–535

Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y,

Rouze P, Rombauts S (2002) PlantCARE, a database of plant

cis-acting regulatory elements and a portal to tools for in silico

analysis of promoter sequences. Nucleic Acids Res 30:325–327

Nagar S, Pedersen TJ, Carrick KM, Hanley-Bowdoin L, Robertson D

(1995) A geminivirus induces expression of a host DNA

synthesis protein in terminally differentiated plant cells. Plant

Cell 7:705–719

Ou-Lee TM, Setter TL (1985) Enzyme activities of starch and sucrose

pathways and growth of apical and Basal maize kernels. Plant

Physiol 79:848–851

Park J, Hwang H, Shim H, Im K, Auh CK, Lee S, Davis KR (2004)

Altered cell shapes, hyperplasia, and secondary growth in

Arabidopsis caused by beet curly top geminivirus infection.

Mol Cells 17:117–124

Park J, Hwang HS, Buckley KJ, Park JB, Auh CK, Kim DG, Lee S,

Davis KR (2010) C4 protein of Beet severe curly top virus is a

pathomorphogenetic factor in Arabidopsis. Plant Cell Rep

29:1377–1389

Parsons TR, Takahashi M, Hargrave B (1984) Plankton feeding and

production In: Biological Oceanographic Processes. 3rd Edition.

Pergamon Press, Oxford, pp 119–143

Riou-Khamlichi C, Menges M, Healy JM, Murray JA (2000) Sugar

control of the plant cell cycle: differential regulation of

Arabidopsis D-type cyclin gene expression. Mol Cell Biol

20:4513–4521

Roitsch T, Gonzalez MC (2004) Function and regulation of plant

invertases: sweet sensations. Trends Plant Sci 9:606–613

Roitsch T, Bittner M, Godt DE (1995) Induction of apoplastic

invertase of Chenopodium rubrum by D-glucose and a glucose

analog and tissue-specific expression suggest a role in sink-

source regulation. Plant Physiol 108:285–294

Ruan YL, Jin Y, Yang YJ, Li GJ, Boyer JS (2010) Sugar input,

metabolism, and signaling mediated by invertase: roles in

development, yield potential, and response to drought and heat.

Mol Plant 3:942–955

Stanley J (1991) The molecular determinants of geminivirus patho-

genesis. Semin Virol 2:139–149

Stanley J, Latham JR, Pinner MS, Bedford I, Markham PG (1992)

Mutational analysis of the monopartite geminivirus beet curly

top virus. Virology 191:396–405

Sturm A, Chrispeels MJ (1990) cDNA cloning of carrot extracellular

[beta]-fructosidase and its expression in response to wounding

and bacterial infection. Plant Cell 2:1107–1119

Sugden C, Crawford RM, Halford NG, Hardie DG (1999) Regulation

of spinach SNF1-related (SnRK1) kinases by protein kinases and

phosphatases is associated with phosphorylation of the T loop

and is regulated by 50-AMP. Plant J 19:433–439

Swiech R, Browning S, Molsen D, Stenger DC, Holbrook GP (2001)

Photosynthetic responses of sugar beet and Nicotiana benthami-

ana Domin. infected with beet curly top virus. Physiol Mol Plant

Pathol 58:43–52

Tymowska-Lalanne Z, Kreis M (1998) The plant invertases: phys-

iology, biochemistry and molecular biology. Adv Bot Res

28:71–117

Wang YQ, Wei XL, Xu HL, Chai CL, Meng K, Zhai HL, Sun AJ,

Peng YG, Wu B, Xiao GF, Zhu Z (2008) Cell-wall invertases

from rice are differentially expressed in caryopsis during the

grain filling stage. J Integr Plant Biol 50:466–474

752 J Plant Res (2013) 126:743–752

123

Author's personal copy