ORIGINAL PAPER

Sugars induce anthocyanin accumulation and flavanone3-hydroxylase expression in grape berries

Yanjun Zheng Æ Li Tian Æ Hongtao Liu Æ Qiuhong Pan ÆJicheng Zhan Æ Weidong Huang

Received: 22 October 2008 / Accepted: 20 March 2009 / Published online: 2 April 2009

� Springer Science+Business Media B.V. 2009

Abstract Flavanone 3-hydroxylase (EC 1.14.11.9, F3H)

plays a key role in anthocyanin biosynthesis, and sugars

enhance anthocyanin accumulation and F3H expression in

some other plants. However, information about the rela-

tionship between sugars, anthocyanin accumulation and

F3H expression in grape berries has been little reported.

Present experiment was done with sliced grape berry

system. The optimum fruit developmental stage, sugar

concentration, and incubation time in sugar induction

anthocyanin accumulation and F3H expression were

determined. Mannose and 2-deoxyglucose, glucose analogs

known to be phosphorylated by hexokinase but are poorly

metabolized, obviously induced the anthocyanin accumu-

lation and F3H expression, whereas 3-O-methylglucose

and 6-deoxyglucose, glucose analogs transported inside the

cell but not substrates for hexokinase, did not induce them.

Glucosamine and mannoheptulose, the specific inhibitors

of hexokinase, blocked the activation induced by sugar on

both anthocyanin accumulation and F3H expression.

Keywords Anthocyanin � Flavanone 3-hydroxylase �Sliced grape berry system � Hexokinase � Sugar signaling

Abbreviations

CHS Chalcone synthase

DAF Day after full bloom

F3H Flavanone 3-hydroxylase

PAL Phenylalanine ammonia lyase

RT-PCR Reverse transcription-polymerase chain

reaction

Introduction

Flavonoids are important plant secondary metabolites

playing a key role in defense against pathogens, protection

from UV radiation, and coloration of flowers and fruits

(Koes et al. 1994; Holton and Cornish 1995). Anthocyanins

which belong to a class of flavonoids are predominant pig-

ments in red and black grape berry skins. It has been found

that the quantity and quality of anthocyanins in grape berries

greatly influence the quality of red wines. It is well known

that anthocyanin accumulation in grape berries commences

at veraison and sugar accumulating begins, and continues

throughout berry ripening (Boss et al. 1996). Anthocyanins

are synthesized via the phenylpropanoid and flavonoid

pathways (Holton and Cornish 1995). The genes of Chal-

cone synthase (CHS), chalcone isomerase (CHI), flavanone

3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR),

anthocyanidin synthase (ANS), and UDP-glucose: flavo-

noid 3-O-glucosyltransferase (UFGT) have been cloned

(Sparvoli et al. 1994) and demonstrated to be activated

during grape berry pigmentation. The f3h gene expression

appears to be pivotal in the regulation at the bifurcation of

the anthocyanins and flavonols branches. It catalyzes the

stereospecific hydroxylation of (2S)-flavanones at 3-posi-

tion of the C-ring to (2R, 3R)-dihydroflavonols (Sparvoli

et al. 1994). The accumulation of anthocyanin during fruit

Y. Zheng � L. Tian � H. Liu � Q. Pan � J. Zhan � W. Huang (&)

College of Food Science and Nutritional Engineering,

China Agricultural University, 100083 Beijing, China

e-mail: huanggwd@263.net

Y. Zheng

e-mail: jollyzyj@yahoo.cn

H. Liu

Institute of Geographic Sciences and Natural Resources

Research, Chinese Academy of Sciences, 100101 Beijing, China

123

Plant Growth Regul (2009) 58:251–260

DOI 10.1007/s10725-009-9373-0

berry development requires a complicated interaction

between environmental and developmental factors. The

factors include the type of varieties, light (Moore et al.

2003), temperature (Mori et al. 2005), plant hormone (Ban

et al. 2003; Loreti et al. 2008), sugars (Vitrac et al. 2000) etc.

In plants, sugars involved in almost all the stage of the

plant life cycle, such as seeding germination, photosyn-

thesis, corolla growth and pigmentation, fruit development

and senescence etc. (Weiss 2000; Ohto et al. 2001; Chen

et al. 2006b). Moreover, it was reported that many types of

genes expression could be regulated by sugars (Villadsen

and Smith 2004; Gonzali et al. 2006). The regulatory

function of sugars is not due merely to a simple metabolic

effect (Rolland et al. 2006) or to an osmotic stress (Sol-

fanelli et al. 2006), it has been proved that sugars can also

act as signaling molecules, and possess a hormone-like

signaling function (Moore et al. 2003; Rolland et al. 2006).

Abscisic acid (ABA), jasmonate (JA), ethylene and stress

often cross-talk with sugar signaling pathway (Gazzarrini

and McCourt 2001; Loreti et al. 2008).

There are three sugar sensing systems have been pro-

posed in plants: hexokinase-dependent pathway (Jang et al.

1997; Vitrac et al. 2000); hexokinase-independent pathway

(Xiao et al. 2000); and the sucrose-specific sensing and

signaling pathway (Solfanelli et al. 2006). Hexokinase,

which was proposed as sugar sensor in many plants, is a

dual-function enzyme with both catalytic effects in gly-

colytic pathway and regulatory functions in sugar sensing

and signaling pathway (Xiao et al. 2000; Cho et al. 2006).

It is very important to uncouple the two functions in order

to distinguish different sugar sensing system in plants.

Some approaches have been performed to investigate the

sugar sensing mechanism involved in anthocyanin bio-

synthesis pathway (Vitrac et al. 2000; Solfanelli et al.

2006), and different sugar signaling pathways operate in

different plant species and processes. In Arabidopsis, the

sugar-dependent up-regulation of anthocyanin synthesis

pathway is sucrose specific. It was found that several genes

including pal, c4h, chs, chi, f3h, f3’h, fls, dfr, ldox, ufgt and

transcription factors MYB75/PAP1 were regulated by

sucrose (Teng et al. 2005; Solfanelli et al. 2006). In radish

hypocotyls, sucrose, glucose and fructose could induce the

anthocyanin production and chs, ans expression, whereas

3-O-methylglucose (glucose analog transported inside the

cell but not a substrate for hexokinase) and mannose

(glucose analog known to be phosphorylated by hexokinase

but is poorly metabolized) could not, which suggested a

hexokinase independent pathway (Hara et al. 2003). In

petunia flowers and grape cell suspensions, sugar promoted

the anthocyanin accumulation and chs expression at least

partially via a hexokinase dependent pathway (Moalem-

Beno et al. 1997; Neta-Sharir et al. 2000; Vitrac et al.

2000). Other signaling intermediates, such as Ca2?,

calmodulin, protein kinases, and protein phosphatases etc.,

are concerned with sugar signaling pathway (Martınez-

Noel et al. 2007; Vitrac et al. 2000).

The effect of sugars on genes involved in anthocyanin

biosynthesis and the potential involvement of hexokinase

have already been studied previously in other plant systems

including grape cell suspensions (Neta-Sharir et al. 2000;

Vitrac et al. 2000). However, the induction of sugars on

anthocyanin accumulation in grape berries is limited,

especially on F3H expression at its RNA and protein level.

In the present works, excised berry system was used for the

first time to follow the sugars induction of anthocyanin

accumulation and F3H expression, and different types of

sugars, glucose analogs and hexokinase inhibitors were

used to investigate the sensory mechanism of sugar. Our

results indicated firstly that 70th day after full bloom

(DAF) berries have the highest sensitivity to sugars effect,

and suggested that sugar induced anthocyanin accumula-

tion in grape berries probably via a hexokinase-dependent

pathway. The research also found firstly that sugars can

modulate F3H gene expression by means of controlling its

RNA and protein levels. All these results might provide a

substantial basis for further research on the control of berry

skin color and wine quality.

Materials and methods

Plant materials

Grape berries (Vitis vinifera L. cv. Cabernet Sauvignon)

were harvested every 10 days from a vineyard in the sub-

urbs of Beijing. The freshly harvested berries were selected

on the basis of similar size and the absence of physical

injuries or insect infections. After washing with distilled

water, the berries were pre-cooled to 25�C. The pretreat-

ments were performed as follow. All the chemicals were

purchased from Sigma (St. Louis, MO 63178, USA) unless

otherwise noted.

The incubation of berry tissues in the mediums

containing sugars

The experiment was performed according to Wen et al.

(2005) with slight modification. The equilibrium buffer

contained 50 mM Mes-Tris [Mes, 2-(N-morpholino) ethane

sulfonic acid; Tris, tris(hydroxymethyl)-amino methane]

(pH 5.5), 1 mM EDTA (ethylenediamine tetraacetic acid),

5 mM ascorbic acid, 5 mM CaCl2, 1 mM MgCl2 and

200 mM mannitol. The grape berries at 70th day after full

bloom (DAF; except when studying the sensitivity of grape

berries at different growth stages to sugars, where the grape

252 Plant Growth Regul (2009) 58:251–260

123

berries at 20, 50, 70, 100th DAF were analyzed) were

sliced into small discs of 0.1 cm thickness, and the disc

tissues were immediately immersed in the equilibrium

buffer for 30 min. The equilibrated tissues of 30 g were

then placed in a 200 ml Erlenmeyer flask containing 90 ml

of the incubation buffer that was composed of the equi-

librium buffer with 100 mM glucose, 100 mM fructose or

150 mM sucrose (except when analyzing the variation of

anthocyanin products in respond to different sugar con-

centrations, where 5, 50, 100, 150, 200 mM of glucose,

fructose and sucrose were used). A pressure of about

60 kPa for 15 min was used for the infiltration of sugars.

The flasks were gently shaken at 25�C for 1 h (sucrose for

2 h, except when analyzing the time course where the pre-

incubation period ranged from 0.5 to 4 h).

In order to investigate the effect of glucose analogs, the

equilibrated tissues (30 g) were incubated in the incubation

mediums that contains 100 mM 3-O-methylglucose, 50 mM

L-glucose, 10 mM 6-deoxyglucose, 2 mM 2-deoxyglucose,

100 mM D-mannose, respectively, by gently shaking for 1 h

at 25�C.

For studying the function of the inhibitors of hexokinase,

the equilibrated tissues (30 g) were preincubated in 90 ml

of the incubation mediums that consist of the equilibrium

buffer with 5 mM mannoheptulose, 100 mM glucosamine

hydrochloride, respectively, by gently shaking for 1 h at

25�C, glucose was then added into pre-incubation mediums.

After the incubation by gently shaking for 1 h at 25�C, at

the end, the incubation mediums were removed and the

tissues were washed three times with double distilled water

and then frozen in liquid nitrogen and stored at -80�C until

use. Each treatment contained three independent replicates.

Measurement of total anthocyanin concentration

Total anthocyanin concentration was measured according

to the method of Ubi et al. (2006) with slight modification.

Berry tissues were ground in liquid nitrogen and extracted

using 1% HCl in methanol with shaking for 4 h in a dark

room. The extracts were centrifuged at 15,0009g for

15 min. After dilution of the extract to 1/10 with a 0.2 M

sodium acetate hydrochloric acid buffer, pH 1.0, absor-

bance at 530 nm was measured with a spectrophotometer

(UV-1600, Shimadzu, Kyoto, Japan). The amount of

anthocyanin concentration was expressed as a milligram of

malvidin-3-glucoside (Extrasynthese, France) equivalents

per gram of fresh berry weight. Mean values were obtained

from three independent replicates.

Sugars quantification

Sugars were extracted from grape berries with double

distilled water and analyzed by HPLC (Shimadzu-10Avp,

Japan). The HPLC system was carried out at 45�C on a

NUCLEOSIL- NH2 column [250 mm 9 4.6 mm (5um),

macherey-nagel, Germany], and was monitored using a

RID-10A detector (Shimadzu, Japan). 75% acetonitrile

were used as an eluent at a flow rate of 1 ml min-1. Glu-

cose, fructose and sucrose were identified by comparing

their retention time with standards, and quantified by peak

area on the chart.

Protein extraction and western blot

Total proteins were extracted according to Chen et al.

(2006a) with modification. The extraction buffer consisted

of 50 mM Tris–HCl (pH 8.9), 2% (w/v) SDS, 5 mM

ascorbic acid, 5 mM EDTA, 1 mM PMSF, 14 mmol l-1

b-mercaptoethanol and 0.15% (w/v) PVP. The separation

of the extracted proteins was performed by SDS–PAGE in

a 12% polyacrylamide gel as described by Laemmli (1970).

The identical amount of protein (3 lg) was loaded per lane.

After electrophoresis, the proteins were electro-transferred

to nitrocellulose (0.45 mm, Amersham LIFE SCIENCE)

using a transfer apparatus (Bio-Rad) according to Isla et al.

(1998). For western blot analysis, immunological detection

of proteins on the NC membrane was carried out using a

primary polyclonal F3H antibody in a 1/1,000 dilution and

alkaline phosphatase conjugated anti-rabbit IgG antibody

from goat (Sigma, St. Louis; 1/500 dilution) as a secondary

antibody at 25�C. Following that, the membrane was

stained with 10 ml of 5- bromo-4-chloro-3-indolyl phos-

phate/nitro blue tetrazolium (BCIP/NBT) in the dark, and

the reaction was terminated by double distilled water. The

intensity of immunoblotting signal was determined by

densitometer.

Isolation of total RNA

Total RNA was isolated from the berry tissues with the

method described by Wen et al. (2005) with slight modi-

fications. All steps were performed at 4�C. The berry

tissues of 1 g was ground in liquid nitrogen and transferred

into 2 ml washing buffer of 0.1 mol l-1 Tris–boracic acid

(pH 7.4), 0.35 mol l-1 sorbitol, 10% PEG6000 (w/v), 2%

bmercaptoethanol (v/v). After centrifugation at 12,0009g

for 8 min, the extraction buffer of 2 ml containing

0.1 mol l-1 Tris–boracic acid (pH 7.4), 1.4 mol l-1 NaCl,

0.02 mol l-1 EDTA and 2% cetyltrimethyl ammonium

bromide (CTAB) was added and rest for 20 min at 55�C.

Then 200 ml potassium acetate of 5 mol l-1, 200 ml eth-

anol and 2 ml chloroform were added. After centrifugation

at 12,0009g for 10 min, 1/3 vol 10 mol l-1 LiCl and 4/5

vol isopropylalcohol were added before centrifugation at

15,0009g. The pellet was dried and then re-suspended in

0.5 ml diethylpyrocarbamate (DEPC)-treated water, and

Plant Growth Regul (2009) 58:251–260 253

123

0.5 ml water-saturated phenol was added. After centrifu-

gation at 15,0009g for 15 min, 0.5 ml chloroform/

isoamylalcohol was added before centrifugation at

15,0009g. Total RNA was then precipitated over night

after addition of 1/3 vol 10 mol l-1 LiCl. After centrifu-

gation (15,0009g, 30 min), the pellet was washed in 75%

ethanol and re-suspended in DEPC water. RNA concen-

tration was determined by absorbance at 260 nm, and

purity was established with a 260/280 ratio.

RT-PCR analysis

The mRNA expression patterns of f3h were examined by

semiquantitative reverse transcription polymerase chain

reaction (RT-PCR), and Actin1 was used as an internal

control. According to published sequences of grape (Gen-

Bank accession nos. X75965 and AY680701), gene-

specific primers for f3h (forward: 50-ATCGTTTCCAGCC

ATCT-30; reverse: 50-GTCTTTCCGCCATCC-30) and

Actin1 (forward: 50-CTGGATTCTGGTGATG-30, reverse:

50-AGGAGCTCTTTGC-30) were used in RT-PCR. The

expected sizes of the PCR products were 389 and 247 bp.

For RT-PCR, first-strand cDNA was synthesized from

1 mg of total RNA in a volume of 20 ll containing 20 mM

Tris–HCl, pH 8.3, 100 mM KCl, 2.5 mM dNTP, 20 units

of RNase inhibitor, 5 mM MgCl2, 5 units of AMV reverse

transcriptase, 2.5 pmol of oligo dT (15) for 45 min at 42�C.

One micro liter of the first-strand solution was used for

PCR reaction in a total volume of 50 ll with 20 mM Tris–

HCl, pH 8.3, 100 mM KCl, 2.5 mM dNTP, 5 units of Taq

DNA polymerase (TaKaRa), 5 mM MgCl2, 10 pmol of

each gene-specific amplification primer. PCR was carried

out with an initial heat action step at 94�C for 5 min, and

amplifications were achieved through 31 cycles at 94�C for

30 s, 52�C for 30 s, and 72�C for 45 s, with a final

extension at 72�C for 10 min. The amplified products were

separated on 1.5% agarose gel.

Results

Glucose, fructose and sucrose enhanced the

anthocyanin accumulation, and the enhancement was

dose-dependent but independent of osmotic effect

In order to investigate whether the osmotic effect would

relate to the effect of sugars on anthocyanin accumulation,

the non-metabolized mannitol, which are not taken up by

the cells was used to create the osmotic pressure gradient in

the system. Grape berries at 70th DAF were incubated by

mannitol with the concentration of 5, 50, 100, 150,

200 mM for 1 h. As shown in Fig. 1, increased osmotic

pressure had scarce effect on the amounts of anthocyanin in

grape berries; hereby the osmotic effect could be excluded

to the effect of sugars on anthocyanin accumulation under

our condition.

Variations in anthocyanin production along with differ-

ent sugar concentrations were shown in Fig. 1. Grape

berries at 70th day after full bloom (DAF) were incubated

by glucose, fructose and sucrose with the concentration of 5,

50, 100, 150, 200 mM for 1 h (sucrose for 2 h). The results

showed that anthocyanin accumulation could be induced by

sugars, and the induction was dose dependent. As shown in

Fig. 1, there is no apparent difference in anthocyanin

Fig. 1 Sugar induction of

anthocyanin accumulation at

different concentrations. Grape

berry tissues were incubated

with 5, 50, 100, 150, 200 mM

mannitol, glucose, fructose,

sucrose, respectively for 1 h

(sucrose for 2 h). The curvesindicated sugars level in grape

berries in response to different

treatment (Mat, mannitol; Glc,

glucose; Fru, fructose; Suc,

sucrose). Values are given as

means and standard errors of

three samples. The same

alphabet represents no

significant difference according

to Duncan’s multiple range tests

at P less than 0.05 levels

254 Plant Growth Regul (2009) 58:251–260

123

products of grape berries when incubated at low sugar level

of 5 mM (except glucose), but higher sugar levels, from

50 mM onwards, suffice to increase the anthocyanin pro-

ductions significantly. Maximum value of anthocyanin

products was attained with 100 mM either glucose or

fructose, or 150 mM sucrose. The levels of glucose, fruc-

tose, and sucrose in grape berry tissues after sugar treatment

were represented as the curves in Fig. 1. Glucose and

fructose in tissues increased along with the corresponding

sugar treatments, and the higher concentrations applied, the

higher levels of sugar appeared in the tissues. Interestingly,

glucose and fructose also increased sharply after sucrose

treatment, and the fructose rose faster than glucose.

Sugars induction was fruit developmental stage

dependent

The sensitivity of grape berries at different growth stages to

sugars were detected (Fig. 2). Grape berries at 20th DAF

(young fruit stage), 50th DAF (developing stage), 70th DAF

(veraison stage), 100th DAF (mature stage) were incubated

with glucose (100 mM), fructose (100 mM) and sucrose

(150 mM), respectively. Figure 2 reveals a correlation

between the sensitivity to sugars and fruit development

stage. Sugar-treated grape berries of most stages (except

developing stage) showed a higher concentration of

anthocyanin accumulation, and the grape berries at the stage

of veraison have the highest sensitivity, about 1.4–1.6 fold

compare with the control. Therefore the grape berries at

70th DAF were selected as materials for experiments below.

Time course studies on anthocyanin accumulation

and F3H expression induced by sugars

Time course studies on sugar enhancing the anthocyanin

accumulation in grape berries

Grape berries were incubated by glucose (100 mM), fruc-

tose (100 mM), or sucrose (150 mM) for 0.5, 1, 2, 4 h.

Figure 3 showed that anthocyanin amounts increased sig-

nificantly after incubated with sugars, and reached

maximum at 1 h (glucose and fructose) and 2 h (sucrose),

respectively. The maximum of anthocyanin amounts were

maintained at the similar level till the end of incubation at

4 h. The anthocyanin contents of sugar-treated berries were

approximately 1.2–1.4 mg malvidin-3-O-glucoside g-1

fresh weight at the maximum while 0.8–0.9 mg malvidin-

3-O-glucoside g-1 fresh weight about that in control

berries.

Sugars elevated F3H by modulating its protein levels

In order to determine the effect of sugars on the protein

level of F3H, western blot analysis was performed. A

41 kDa peptide was specifically detected with F3H poly-

clonal antibody on SDS–PAGE gel of the berry crude

extract. As shown in Fig. 4, the amounts of F3H protein

reached the peak at 1 h after glucose treated and at 2 h

after sucrose treated, then declined. The F3H protein in

grape berries of fructose-treated presented an upwards

tendency along with the incubation time.

Fig. 2 The sensitivity to sugars

of grape berry tissues at

different growth stages. Grape

berries at 20th, 50th, 70th, 100th

DAF were incubated with

glucose (100 mM), fructose

(100 mM), sucrose (150 mM)

for 1 h (sucrose for 2 h). The

tissues incubated with 200 mM

mannitol were taken as control.

The curves indicated that sugars

level in vivo after incubation

with different sugars. Glc:

glucose; Fru: fructose; Suc:

sucrose. The plotted data and

error bars indicate the means

and variations of three samples.

The same alphabet represent no

significantly different at the

0.05 level according to

Duncan’s multiple range test

Plant Growth Regul (2009) 58:251–260 255

123

Sugars elevated f3h by modulating its RNA levels

To evaluate the effect of sugars on the f3h mRNA level, the

response to glucose, fructose and sucrose, at different

incubation time were analyzed by RT-PCR. As shown in

Fig. 5, the f3h gene expression was dramatically enhanced

by sugars treatment, and the f3h RNA level started to

increase until reached a maximum at 1 h (treated by glu-

cose and fructose) and at 2 h (treated by sucrose), and then

decreased immediately, after 4 h of incubation, f3h RNA

level was almost the same to the control.

Effect of glucose analogs on anthocyanin accumulation

and F3H expression

According to previous reports on the functions of

glucose analogs, 100 mM 3-O-methylglucose, 10 mM

6-deoxyglucose, 50 mM L-glucose, 2 mM 2-deoxyglucose

and 100 mM mannose were used to assess the role of

hexokinase in sugar induced anthocyanin biosynthetic

pathway. We firstly tested the effect of the 3-O-methyl-

glucose, 6-deoxyglucose (glucose analogs transported

inside the cell but not a substrate for hexokinase) and

L-glucose (which is not recognized by hexose transporters

and is often used as osmotica). The anthocyanin contents

were approximately 0.7–0.8 mg malvidin-3-O-glucoside

g-1 fresh weight and slight declines compared to the

control (0.87 mg malvidin-3-O-glucoside g-1 fresh

weight). Then we examined the effect of mannose and

Fig. 3 Time course of sugars-

induced anthocyanin

accumulation. Grape berries

were incubated by glucose

(100 mM), fructose (100 mM),

or sucrose (150 mM) for 0.5,

1, 2, 4 h. The tissues incubated

with 200 mM mannitol were

taken as control. The curvesindicated the sugar levels in

vivo after treated by different

sugars. Glc: glucose; Fru:

fructose; Suc: sucrose. The

plotted data and error barsindicate the means and

variations of three samples. The

same letters are not significantly

different at the 0.05 level

according to Duncan’s multiple

range test

Fig. 4 Effect of glucose, fructose and sucrose on the amounts of F3H

protein in grape berries. Grape berries were incubated by glucose

(100 mM), fructose (100 mM), or sucrose (150 mM) for 0.5, 1, 2,

4 h. A 41 kDa peptide was specifically detected with F3H polyclonal

antibody by western blot analysis. The arrows indicate the position of

F3H protein. Glc: glucose; Fru: fructose; Suc: sucrose

Fig. 5 Effect of glucose, fructose and sucrose on the RNA level of

f3h gene in grape berries. Grape berries were incubated by glucose

(100 mM), fructose (100 mM), or sucrose (150 mM) for 0.5, 1, 2,

4 h. Actin1 was used as internal control gene to ensure identity in the

amounts of RNA. The expected sizes of the PCR products were

389 bp. Glc: glucose; Fru: fructose; Suc: sucrose

256 Plant Growth Regul (2009) 58:251–260

123

2-deoxyglucose, glucose analogs known to be phosphory-

lated by hexokinases but is poorly metabolized. As shown

in Fig. 6a, Mannose and 2-deoxyglucose enhanced the

anthocyanin accumulations obviously with the value of

1.16 mg malvidin-3-O-glucoside g-1 fresh weight (Man-

nose) and 1.01 mg malvidin-3-O-glucoside g-1 fresh

weight (2-deoxyglucose). In addition, we tested F3H pro-

tein level in response to sugar analogs. Western blot

analysis indicated that there was no significant difference on

the protein level of F3H among the control, 3-O-methyl-

glucose, 6-deoxyglucose and L-glucose-treated grape

berries. On the contrary, mannose and 2-deoxyglucose

notably increased the amounts of F3H protein (Fig. 6b).

The f3h RNA level was also measured. As shown in Fig. 6c,

3-O-methylglucose, 6-deoxyglucose, and L-glucose had no

apparent effect on the induction of the RNA level expres-

sion of f3h whereas mannose and 2-deoxyglucose strongly

enhanced it.

Glucosamine and mannoheptulose abolished

the promotive effect of glucose

To evaluate the role of hexokinase in our model, we tried to

determine the effect of specific hexokinase inhibitors such

as glucosamine and mannoheptulose. As shown in Fig. 7a,

the addition of glucose could not increase the amounts of

anthocyanin in grape berries pre-treated by glucosamine or

mannoheptulose. Refer to the F3H expression, both the

western blot and RT-PCR analysis indicated that

glucosamine and mannoheptulose could block the sugar

induction of F3H expression.

Discussion

Many studies have shown that the anthocyanin accumula-

tion in plants can be induced by sugar; of which sugar seem

to be not only general carbohydrate sources, but also act as

signal molecule to activate/repress the reactions (Mita et al.

1997; Weiss 2000). In our experiment, glucose, fructose

and sucrose could induce the anthocyanin accumulation in

grape berries with the optimum concentration 100 mM

(glucose and fructose) or 150 mM (sucrose), but increasing

concentration of mannitol (from 5 to 200 mM) was not

sufficient to induce it (Fig. 1). In addition, 3-O-methyl-

glucose (100 mM) and L-glucose (50 mM) had no effect

either on anthocyanin products or F3H expression whereas

mannose and 2-deoxyglucose obviously induced them

(Fig. 6). All these indicated that the attained effect of

sugars on anthocyanin accumulation in grape berries was

sugar specific and at least partially independent of osmotic

effect and metabolism effect.

A sucrose specific signaling pathway has been proposed

by many studies. In Arabidopsis, the sugar induction effect

on anthocyanin accumulation is established via a sucrose

specific pathway (Teng et al. 2005; Solfanelli et al. 2006),

and the disaccharide analogs palatinose and turanose were

used to illustrate the sucrose-specific signaling pathway in

Fig. 6 The effect of different

glucose analogs on anthocyanin

accumulation (a), F3H protein

level (b) and RNA level

expression (c). Grape berries

were treated by different

glucose analogs for 1 h.

Western blot and RT-PCR were

performed to investigate the

F3H protein level and RNA

level expression induced by

sugars, respectively. Lane 1–7:

CK, Glucose, 6-Deoxyglucose,

3-O-methylglucose, L-Glucose,

2-Deoxyglucose, Mannose. The

arrows indicate the position of

F3H protein. The plotted dataand error bars indicate the

means and variations of three

samples

Plant Growth Regul (2009) 58:251–260 257

123

potato tubers and barley embryos (Yang et al. 2004). But in

our model, the sucrose-specific mechanism did not seem to

be involved. We noticed that sugars enhanced the antho-

cyanin accumulation, at the same time, the concentrations

of corresponding sugar in tissues increased, except sucrose,

which was rarely detected even in sucrose-treated grape

berries (Figs. 1, 2, 3). However, the contents of hexose

(glucose, fructose) exhibited a relative increase along with

the sucrose incubation, and the contents of fructose rose

faster than glucose, this probably due to the conjunct effect

of invertase which hydrolyses sucrose into fructose and

glucose, and sucrose synthase which converts sucrose and

UDP into fructose and UDP-glucose (Koch 2004). More-

over, glucose and fructose enhanced the anthocyanin

accumulation similar to sucrose (Figs. 1, 2, 3), leading to

the hypothesis that the sensing of hexoses was dominant in

sugar response pathway in our system.

Many studies have shown that hexoses act as signal

molecules in higher plants (Vitrac et al. 2000), and in most

cases the phosphorylation of hexose by hexokinase is

required to initiate the hexoses signal transduction pathway

(Vitrac et al. 2000). In order to investigate the role of

hexokinase in hexose signaling pathway, we have studied

the effect of glucose analogs on anthocyanin accumulation.

The results showed that neither 3-O-methylglucose nor 6-

deoxyglucose could induce the anthocyanin accumulation

whereas mannose and 2-deoxyglucose had positive effect

on anthocyanin accumulation (Fig. 6). Therefore, it might

be concluded that the sugar sensing in our system does not

occur before hexokinase, and only analogs that are sub-

strates for hexokinase were able to modulate the

anthocyanin accumulation.

As shown in Fig. 7, both glucosamine and mannohep-

tulose could almost completely abolish the anthocyanin

accumulation induced by sugars, which further supported

the involvement of hexokinase in sugar signaling pathway

(Neta-Sharir et al. 2000; Vitrac et al. 2000). Other pub-

lished evidence also indicated that hexokinase played a

regulatory role in sugar sensing pathway by using the

antisense and overexpression technologies (Jang et al.

1997, Sun et al. 2006). It was found that antisense plants

with reduced expression of hexokinase were hyposensitive

to glucose and 2-deoxyglucose whereas enhanced sensi-

tivity to these sugars was observed in hexokinase-

overexpressing plants.

Anthocyanin accumulation was induced by sugars; this

apparently correlates with the gene expression involved in

anthocyanin biosynthesis. Many anthocyanin synthesis

structural and regulatory genes in grape have been cloned

(Sparvoli et al. 1994) and some of them were demonstrated

to be induced by sugars (Neta-Sharir et al. 2000; Solfanelli

et al. 2006). CHS and ANS expression were reported to be

Fig. 7 The effect of

glucosamine and

mannoheptulose on the

anthocyanin accumulation (a),

F3H protein level (b) and RNA

level expression (c). Grape

berry tissues were pre-incubated

by glucosamine (100 mM) or

mannoheptulose (5 mM) for

1 h, and then glucose was added

into the pre-incubation mediums

for 1 h. Lane 1–4: CK, Glucose,

Glucosamine ? Glucose,

Mannoheptulose ? Glucose.

The arrows indicate the position

of F3H protein. The plotted dataand error bars indicate the

means and variations of three

samples

258 Plant Growth Regul (2009) 58:251–260

123

induced by sugars in Arabidopsis (Solfanelli et al. 2006),

soybean leaves (Sadka et al. 1994) and grape cells sus-

pension (Vitrac et al. 2000). DFR and F3H expression was

also activated by sucrose in Arabidopsis (Solfanelli et al.

2006; Loreti et al. 2008). Interestingly, petunia and Ara-

bidopsis CHS genes possess sucrose boxes in the 50-flanking regions. These sucrose boxes were found in the

upstream region of sporamin and b-amylase genes, which

are induced by sucrose (Tsukaya et al. 1991). In present

experiment, sugars could induce F3H expression along

with the incubation time (Fig. 4); neither 3-O-methylglu-

cose nor 6-deoxyglucose had any activation on F3H

expression at either RNA or protein level, whereas man-

nose and 2-deoxyglucose induced them obviously (Fig. 6b,

c). Glucosamine and mannoheptulose, specific inhibitors of

hexokinase, blocked the expression of F3H (Fig. 7b, c). All

these results suggested that sugars could activate F3H by

means of controlling the expression of F3H RNA level and

protein level via a hexokinase dependent pathway.

In conclusion, sugars induced the anthocyanin accu-

mulation in grape berries; the induction is not simply due to

metabolic effect or osmotic effect; sugars can be act as

signal molecules and able to initiate a series of signal

cascade; sugar induced the F3H expression at its RNA and

protein level; sugar sensing in our systems is via a hexo-

kinase-dependent pathway, and the hexokinase act as a

sugar sensor. On the basis of sliced berry system, it is

investigated for the first time, to our knowledge that sugars

induce anthocyanin accumulation in grape berries which is

dependent on development stage and the response of F3H

protein and RNA level to sugars in grape berries. Further

studies on sugar signaling pathway in grape berries are

suggested.

Acknowledgments This research was supported by major program

of Beijing Municipal Science & Technology Commission (No.

D07060500160701).

References

Ban T, Ishimaru M, Kobayashi S, Shiozaki S, Goto-Yamamoto N,

Horiuchi S (2003) Abscisic acid and 2,4-dichlorohenoxyacetic

acid affect the expression of anthocyanin biosynthetic pathway

genes in ‘Kyoho’ grape berries. J Hortic Sci Biotechnol 78:586–

589

Boss PK, Davies C, Robinson SP (1996) Analysis of the expression of

anthocyanin pathway genes in developing vitis vinifera 1 cv

shiraz grape berries and the implications for pathway regdation.

Plant Physiol 111:1059–1066

Chen JY, Wen PF, Kong WF, Pan QH, Zhan JC, Li JM, Wan SB,

Huang WD (2006a) Effect of salicylic acid on phenylpropanoids

and phenylalanine ammonia-lyase in harvested grape berries.

Postharvest Biol Technol 40:64–72. doi:10.1016/j.postharvbio.

2005.12.017

Chen Y, Ji FF, Xie H, Liang JS, Zhang JH (2006b) The regulator of

G-protein signaling proteins involved in sugar and abscisic acid

signaling in Arabidopsis seed germination. Plant Physiol

140:302–310. doi:10.1104/pp.105.069872

Cho J, Ryoo N, Ko S, Lee SK, Lee J, Jung KH, Lee YH, Bhoo SH,

Winderickx J, An G, Hahn TR, Jeon JS (2006) Structure,

expression, and functional analysis of the hexokinase gene

family in rice (Oryza sativa L.). Planta 224:598–611. doi:

10.1007/s00425-006-0251-y

Gazzarrini S, McCourt P (2001) Genetic interactions between ABA,

ethylene and sugar signalling pathways. Curr Opin Plant Biol

4:387–391

Gonzali S, Loreti E, Solfanelli C, Novi G, Alpi A, Perata P (2006)

Identification of sugar-modulated genes and evidence for in vivo

sugar sensing in Arabidopsis. J Plant Res 119:115–123. doi:

10.1007/s10265-005-0251-1

Hara M, Oki K, Hoshino K, Kuboi T (2003) Enhancement of

anthocyanin biosynthesis by sugar in radish (Raphanus sativus)

hypocotyls. Plant Sci 164:259–265. doi:10.1016/S0168-9452(02)

00408-9

Holton TA, Cornish EC (1995) Genetics and biochemistry of

anthocyanin biosynthesis. Plant Cell 7:1071–1083

Isla MI, Vattuone MA, Sampietro AR (1998) Essential group at the

active site of Frapaeolum invertase. Phytochemistry 47:1189–

1193. doi:10.1016/S0031-9422(97)00757-7

Jang JC, Leon P, Zhou L, Sheen JS (1997) Hexokinase as a sugar

sensor in higher plants. Plant Cell 9:5–19

Koch K (2004) Sucrose metabolism: regulatory mechanisms and

pivotal roles in sugar sensing and plant development. Curr Opin

Plant Biol 7:235–246. doi:10.1016/j.pbi.2004.03.014

Koes R, Quattrocchio R, Mol J (1994) The flavonoid biosynthetic

pathway in plants: function and evolution. Bioessays 16:123–

132. doi:10.1002/bies.950160209

Laemmli UK (1970) Cleavage of structural protein during the

assembly of the head of bacteriophage T4. Nature 227:680–685.

doi:10.1038/227680a0

Loreti E, Povero G, Novi G, Solfanelli C, Alpi A, Perata P (2008)

Gibberellins, jasmonate and abscisic acid modulate the sucrose-

induced expression of anthocyanin biosynthetic genes in Ara-bidopsis. New Phytol 4:1–13

Martınez-Noel G, Nagaraj V, Calo G, Wiemken A, Pontis HG (2007)

Sucrose regulated expression of a Ca2t-dependent protein kinase

(TaCDPK1) gene in excised leaves of wheat. Plant Physiol

Biochem 45:410–419. doi:10.1016/j.plaphy.2007.03.004

Mita S, Murano N, Akaike M, Nakamura K (1997) Mutants of

Arabidopsis thaliana with pleiotropic effects on the expression of

the gene for beta-amylase and on the accumulation of anthocy-

anin that are inducible by sugars. Plant J 11:841–851. doi:

10.1046/j.1365-313X.1997.11040841.x

Moalem-Beno D, Tamari G, Leitner-Dagan Y, Borochov A, Weiss D

(1997) Sugar-dependent gibberellin-induced chalcone synthase

gene expression in petunia corollas. Plant Physiol 113:419–424

Moore B, Zhou L, Rolland F, Hall Q, Cheng WH, Liu YX, Hwang I,

Jones T, Sheen J (2003) Role of the Arabidopsis glucose sensor

HXK1 in nutrient, light, and hormonal signaling. Science

300:332–336. doi:10.1126/science.1080585

Mori K, Sugaya S, Gemma H (2005) Decreased anthocyanin

biosynthesis in grape berries grown under elevated night

temperature condition. Sci Hortic 105:319–330. doi:10.1016/

j.scienta.2005.01.032

Neta-Sharir I, Shoseyov O, Weiss D (2000) Sugars enhance the

expression of gibberellin-induced genes in developing petunia

flowers. Physiol Plant 109:196–202. doi:10.1034/j.1399-3054.

2000.100212.x

Ohto M, Onai K, Furukawa Y, Aoki E, Araki T, Nakamura K (2001)

Effects of sugar on vegetative development and floral transition

in Arabidopsis. Plant Physiol 127:252–261. doi:10.1104/pp.127.

1.252

Plant Growth Regul (2009) 58:251–260 259

123

Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and

signaling in plants: conserved and novel mechanisms. Annu Rev

Plant Biol 57:675–709. doi:10.1146/annurev.arplant.57.032905.

105441

Sadka A, Dewald DB, May GD, Park WD, Mullet JE (1994)

Phosphate modulates transcription of soybean VspB and other

sugar inducible genes. Plant Cell 6:737–749

Solfanelli C, Poggi A, Loreti E, Alpi A, Perata P (2006) Sucrose-

Specific induction of the anthocyanin biosynthetic pathway in

Arabidopsis. Plant Physiol 140:637–646. doi:10.1104/pp.105.

072579

Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C (1994)

Cloning and molecular analysis of structural genes involved in

flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.).

Plant Mol Biol 24:743–755. doi:10.1007/BF00029856

Sun JY, Chen YM, Wang QM, Chen J, Wang XC (2006) Glucose

inhibits the expression of triose phosphate/phosphate transloca-

tor gene in wheat via hexokinase-dependent mechanism. Int J

Biochem Cell Biol 38:1102–1113

Teng S, Keurentjes J, Bentsink L, Koornneef M, Smeekens S (2005)

Sucrose-specific induction of anthocyanin biosynthesis in Ara-bidopsis requires the MYB75/PAP1 gene. Plant Physiol

139:1840–1852. doi:10.1104/pp.105.066688

Tsukaya H, Ohshima T, Naito S, Chino M, Komeda Y (1991) Sugar

dependent expression of the CHS-A gene for chalcone synthase

from petunia in transgenic Arabidopsis. Plant Physiol 97:1414–

1421. doi:10.1104/pp.97.4.1414

Ubi BE, Honda C, Bessho H, Kondo S, Wada M, Kobayashi S,

Moriguchi T (2006) Expression analysis of anthocyanin biosyn-

thetic genes in apple skin: effect of UV-B and temperature. Plant

Sci 170:571–578. doi:10.1016/j.plantsci.2005.10.009

Villadsen D, Smith S (2004) Identification of more than 200

glucoseresponsive Arabidopsis genes none of which responds

to 3-O-methylglucose or 6-deoxyglucose. Plant Mol Biol

55:467–477. doi:10.1007/s11103-004-1050-0

Vitrac X, Larronde F, Krisa S, Decendit A, Deffieux G, Merillon JM

(2000) Sugar sensing and Ca2?-calmodulin requirement in Vitis

vinifera cells producing anthocyanins. Phytochemistry 53:659–

665. doi:10.1016/S0031-9422(99)00620-2

Weiss D (2000) Regulation of flower pigmentation and growth:

multiple signaling pathways control anthocyanin synthesis in

expanding petals. Physiol Plant 110:152–157. doi:10.1034/

j.1399-3054.2000.110202.x

Wen PF, Chen JY, Kong WF, Pan QH, Wan SB, Huang WD (2005)

Salicylic acid induced the expression of phenylalanine ammonia-

lyase gene in grape berry. Plant Sci 169:928–934. doi:

10.1016/j.plantsci.2005.06.011

Xiao W, Sheen J, Jang JC (2000) The role of hexokinase in plant

sugar signal transduction and growth and development. Plant

Mol Biol 44:451–461. doi:10.1023/A:1026501430422

Yang ZP, Zhang L, Diao FQ, Huang MJ, Wu NH (2004) Sucrose

regulates elongation of carrot somatic embryo radicles as a

signal molecule. Plant Mol Biol 54:441–459. doi:10.1023/

B:PLAN.0000036375.40006.d3

260 Plant Growth Regul (2009) 58:251–260

123