ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance

ABF2, an ABRE-binding bZIP factor, is an essential componentof glucose signaling and its overexpression affects multiplestress tolerance

Sunmi Kim, Jung-youn Kang, Dong-Im Cho, Ji Hye Park and Soo Young Kim*

Kumho Life and Environmental Science Laboratory, 1 Oryong-dong, Buk-gu, Gwangju 500-712, South Korea

Received 5 May 2004; accepted 29 June 2004.*For correspondence: (fax þ82 62 972 5085; e-mail [email protected]).

Summary

Phytohormone abscisic acid (ABA) regulates stress-responsive gene expression during vegetative growth,

which is mediated largely by cis-elements sharing the ACGTGGC consensus. Although many transcription

factors are known to bind the elements in vitro, only a few have been demonstrated to have in vivo functions

and their specific roles in ABA/stress responses are mostly unknown. Here, we report that ABF2, an ABF

subfamily member of bZIP proteins interacting with the ABA-responsive elements, is involved in ABA/stress

responses. Its overexpression altered ABA sensitivity, dehydration tolerance, and the expression levels of

ABA/stress-regulated genes. Furthermore, ABF2 overexpression promoted glucose-induced inhibition of

seedling development, whereas its mutation impaired glucose response. The reduced sugar sensitivity was

not observed with mutants of two other ABF family members, ABF3 and ABF4. Instead, these mutants

displayed defects in ABA, salt, and dehydration responses, whichwere not observedwith the abf2mutant. Our

data indicate distinct roles of ABF family members: whereas ABF3 and ABF4 play essential roles in ABA/stress

responses, ABF2 is required for normal glucose response. We also show that ABF2 overexpression affects

multiple stress tolerance.

Keywords: abscisic acid, ABA-responsive element, bZIPprotein,ABF/AREB, stress response, glucose signaling.

Introduction

The phytohormone abscisic acid (ABA) controls various

aspects of plant growth throughout development

(Finkelstein et al., 2002; Zeevaart and Creelman, 1988). It

prevents embryos from precocious germination, promotes

seedling growth under normal conditions (Cheng et al.,

2003), and regulates seed maturation. During vegetative

growth, its major role is to mediate adaptive responses to

various adverse environmental conditions known as

‘environmental’ or ‘abiotic stresses.’ Its critical role in the

adaptation or acclimation to drought, freezing, and high

salinity is well documented (Schroeder et al., 2001;

Thomashow, 1999; Xiong et al., 2002). Moreover, a num-

ber of studies show that the hormone is required for the

full protection against heat stress. ABA treatments of cul-

tured cells or seedlings promote their heat tolerance (Gong

et al., 1998; Larkindale and Knight, 2002; Robertson et al.,

1994). In contrast, a mutant defective in ABA signaling (i.e.

abi-1) is susceptible to high temperature (Larkindale and

Knight, 2002). ABA also plays an important role in

oxidative stress response, by regulating genes or enzyme

activities involved in the removal of reactive oxygen spe-

cies (e.g. superoxide radical and H2O2) that accumulate

under various stress conditions to cause cellular oxidative

damages (Desikan et al., 2001; Jiang and Zhang, 2002;

Pastori and Foyer, 2002). Although essential for normal

growth and adaptive stress responses, high concentrations

of ABA have inhibitory effect on seed germination and

seedling growth (Himmelbach et al., 1998).

One of the key ABA-dependent processes under various

stress conditions is the regulation of gene expression.

Numerous genes involved in stress responses are up-

or down-regulated by ABA (Bartholomew et al., 1991;

Ramanulu and Bartels, 2002; Shinozaki and Yamaguchi-

Shinozaki, 2000; Wang et al., 1996; Weatherwax et al., 1996).

Promoter analyses of the ABA-regulated genes revealed that

many of the ABA-regulated genes are controlled by the cis-

regulatory elements sharing the (C/T)ACGTGGC consensus

(Busk and Pages, 1998; Hattori et al., 2002; Rock, 2000). The

ª 2004 Blackwell Publishing Ltd 75

The Plant Journal (2004) 40, 75–87 doi: 10.1111/j.1365-313X.2004.02192.x

conserved sequence motif, generally known as ‘ABA-

responsive element (ABRE),’ is a subset of the G-box

sequence (CACGTG) present in the promoter regions of

many light-regulated genes (Giuliano et al., 1988). Numer-

ous basic leucine zipper (bZIP) class proteins (Landschulz

et al., 1988) have been isolated based on their in vitro

interactions with the ABRE or the G-box (Busk and Pages,

1998; Foster et al., 1994; Menkens et al., 1995; Rock, 2000).

However, reports on their specific roles in ABA and/or stress

responses have been scarce.

We and others previously reported a small subfamily of

ABRE-binding bZIP proteins designated as ABFs (i.e. ABF1–

ABF4) or AREBs (i.e. AREB1–AREB3) (Choi et al., 2000; Uno

et al., 2000). The transcription factors are highly homologous

to ABI5 (Finkelstein and Lynch, 2000; Lopez-Molina and

Chua, 2000), which is a genetically identified ABA signaling

component playing an essential role in seed germination

and ABA-triggered post-germination developmental arrest

process (Lopez-Molina et al., 2001). Recent studies show that

ABI5 belongs to a subfamily consisting of embryo-abundant,

ABF-related factors (Bensmihen et al., 2002; Kim et al., 2002).

ABF family members, on the contrary, are expressed mainly

in vegetative tissues, and their expression is induced by

various abiotic stresses (Choi et al., 2000; Uno et al., 2000).

Additionally, their distinct stress-induction patterns suggest

that each ABF may function in different stress response

pathways; i.e. ABF1 in cold signaling, ABF2 and ABF3 in salt

signaling, and ABF4 in cold, salt, and drought signaling

pathways. ABFs can transactivate ABRE-containing reporter

genes. It has been demonstrated that ABA-dependent post-

translational modification, probably phosphorylation, is

required for the maximal transcriptional activity of ABF2

(AREB1) and ABF4 (AREB2) (Uno et al., 2000).

Recently, in vivo analysis of ABF3 and ABF4 functions has

been reported (Kang et al., 2002). Overexpression of ABF3 or

ABF4 in Arabidopsis resulted in ABA hypersensitivity and

enhanced drought tolerance with changes in the expression

levels of a number of ABA- or stress-regulated genes.

However, it remains to be determined whether other ABF

family members, ABF1 and ABF2, are also involved in ABA/

stress responses, and if so, what their specific roles are.

Here, we report ABF2 overexpression phenotypes, which

suggest its role in ABA, stress, and glucose responses. In

addition, we show by mutant analyses that, whereas ABF3

and ABF4 are essential for normal ABA/stress responses,

ABF2 is necessary for seedling growth regulation and

glucose response.

Results

Expression patterns of ABF2

Our previous study showed that ABF2 expression in seed-

lings is induced by ABA and high salt (Choi et al., 2000). To

gain further clues about ABF2 function, we investigated its

tissue-specific expression pattern by determining its RNA

levels in various tissues using coupled reverse transcription

and polymerase reaction (RT-PCR). ABF2 expression was

detected in all tissues under normal growth conditions:

leaves, roots, flowers, and siliques (Figure 1a). Next, we

determined its temporal and spatial expression patterns by

histochemical b-glucuronidase (GUS) staining of the trans-

genic plants transformed with a 2.3-kb ABF2 promoter-GUS

fusion construct. GUS activity was not observed in embryos

from green siliques, but strong activity was detected in the

axes, especially, in radicles and shoot meristem regions of

embryos from dry siliques (Figure 1b, panels a and b). A

similar staining pattern (i.e. strong staining of the emerging

roots and shoots, and relatively weaker staining of hypoco-

tyls) was retained in germinating seedlings (Figure 1b, panel

c). During subsequent seedling growth, GUS activity in the

primary root attenuated, and the highest activity was

detected in lateral roots except the meristem regions and the

elongation zones (Figure 1b, panel d). Rosette leaves also

exhibited GUS activity, especially, in the veins and guard

cells (Figure 1b, panels e and f). Among the reproductive

organs, strong GUS activity was observed in anthers, fila-

ments, stigma, and immature siliques (abscission zone,

valves, replum, and funiculi) (Figure 1b, panels g and h). In

summary, ABF2 promoter was active in the axes of mature

embryos, in most of the vegetative tissues, and in several

reproductive organs, suggesting that ABF2 may function in

many organs throughout development.

Our study described below indicated that ABF2 is

involved in glucose response and that its activity may be

modified by sucrose at the germination/young seedling

stage. We therefore investigated whether its expression is

modulated by sugars at the early stage of growth. Glucose

did not affect ABF2 expression level (data not shown).

However, Figure 1(c) shows that ABF2 RNA level increased

with increasing concentrations of sucrose in the germina-

tion medium, demonstrating the sucrose inducibility of

ABF2 expression. Figure 1(c) also shows that, in contrast

to ABF2, expression of ABF3 and ABF4 was repressed by

sucrose. Sucrose inducibility thus appears to be ABF2-

specific.

Growth phenotypes of 35S-ABF2 plants

To investigate the in vivo functions of ABF2, we generated

transgenic Arabidopsis plants expressing ABF2 under the

control of the strong, cauliflower mosaic virus 35S promoter

(Odell et al., 1985). T3 or T4 homozygous lines were recov-

ered and used for phenotypic analysis (see Experimental

procedures).

As ABA regulates growth, we examined the effects of

ABF2 overexpression on plant growth. 35S-ABF2 plants

expressing ABF2 at high levels germinated and grew slowly

76 Sunmi Kim et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 75–87

compared with wild type plants (Figure 2a–c). As a conse-

quence, bolting of 35S-ABF2 plants was delayed several

days. Growth/maturation after transition to reproductive

stage were also retarded (Figure 2d), and it took longer (up

to approximately 2 weeks) than wild type plants for the 35S-

ABF2 plants to senesce. Despite their slower growth/matur-

ation, 35S-ABF2 plants developed normally and mature

plants exhibited little dwarfism (Figure 2d), indicating that

ABF2 overexpression resulted in retarded growth without

affecting developmental process. The growth retardation of

young 35S-ABF2 seedlings was alleviated significantly

when the growth medium was supplemented with sucrose

(Figure 2e).

ABF2 overexpression affects the expression of ABA- and/or

stress-responsive genes

Expression levels of a number of ABA- or stress-regulated

genes in the wild type and 35S-ABF2 transgenic plants were

compared to investigate the transcriptional regulatory role

of ABF2. As shown in Figure 3(a), the RNA levels of rd29A

(Yamaguchi-Shinozaki and Shinozaki, 1994), chalcone syn-

thase (CHS) (Feinbaum and Ausubel, 1988), and sucrose

synthase1 (SUS1) (Martin et al., 1993) were lower in the

transgenic plants than in wild type plants. The result sug-

gested that expression of the genes, which are normally

upregulated by ABA or abiotic stresses, was repressed by

ABF2. Uno et al. (2000) demonstrated that ABF2 (i.e. AREB2)

needs to be activated by ABA for its maximal transcriptional

activity in a transient assay system. To examine whether

similar post-translational modification is required for nor-

mal ABF2 activity in vivo, we determined the RNA levels of

the same genes under high salt condition. Figure 3(a) shows

that the RNA levels of rd29A, CHS, and SUS1 were higher in

35S-ABF2 plants than in wild type plants when the plants

were treated with 250 mM NaCl solution. Thus, on the con-

trary to its repressive role under normal condition, ABF2

enhanced their expression under the high salt condition,

suggesting that post-translational modification, triggered by

high salt in this case, was necessary for its transcriptional

activation function. However, the ADH1 gene (de Bruxelles

et al., 1996) was expressed at higher levels in 35S-ABF2

plants under both normal and high salt conditions

(Figure 3a). It appears that the regulatory mechanism of

ADH1 expression is different from those of the above ABA-

responsive genes.

Several studies showed that ABA suppresses the

expression of photosynthetic genes in other species

(Bartholomew et al., 1991; Wang et al., 1996; Weatherwax

et al., 1996). Similarly, the rbcS message level of wild type

Arabidopsis seedlings reduced significantly by ABA treat-

ment (Figure 3b), indicating that Arabidopsis rbcS expres-

sion is subject to negative regulation by ABA. To test

whether ABF2 is involved in the process, we determined

the rbcS levels in 35S-ABF2 plants. Figure 3(b) shows that

the rbcS RNA levels in the transgenic plants were lower

than in wild type plants. Thus, ABF2 overexpression

suppressed the rbcS expression, suggesting that ABF2

may be involved in the ABA-regulation of rbcS.

As germination and seedling growth were significantly

inhibited by ABF2 overexpression and inhibition was

(a) L R F S

Actin

ABF2

(c)0% 1% 3% 6%

Sucrose

ABF2

ABF3

ABF4

Actin

(b)

f

g

c d

e h

a

b

Figure 1. Expression patterns of ABF2.

(a) Tissue-specificity of ABF2 expression. ABF2 RNA levels in various tissues

under normal conditions were determined by RT-PCR. L, leaves from 3-week-

old plants; R, roots from 3-week-old plants; F, flowers; S, immature siliques.

(b) Histochemical GUS staining of ABF2 promoter (2.3 kb)-GUS transgenic

plants. a, an embryo from immature silique; b, an embryo from dry seed; c, a

3-day-old seedling; d, root of 2-week-old seedling; e, a 2-week-old seedling; f,

guard cells; g, a flower; i, an immature silique. T2 generation plants were

stained for 18 h.

(c) Sucrose inducibility of ABF2 expression. Wild type (Ler) seeds were

germinated on MS medium containing various concentrations of sucrose for

4 days, and the ABF RNA levels were determined by RT-PCR.

ABF2 is a positive component of glucose signaling 77


relieved by the addition of sucrose to the growth medium

(Figure 2e), we investigated the expression levels of several

genes that are associated with reserve mobilization or energy

metabolism. Most prominent changes were observed with

hexokinase genes. As shown in Figure 3(c), the expression of

HXK1 and HXK2 (Jang et al., 1997) was not detected at all in

35S-ABF2 seedlings germinating on a medium lacking

sucrose, suggesting that ABF2 abolished their expression.

The repression of hexokinase gene expression was not

observed when the germination medium was supplemented

with 1% sucrose (Figure 3c) and at later seedling growth

stages (data not shown). The ABF2 repression of the expres-

sion of HXK genes was thus dependent on the availability of

exogenous sucrose and developmental stage.

ABF2 overexpression affects ABA sensitivity

To investigate whether ABF2 overexpression affected ABA

response, we conducted ABA dose–response analyses of

germination and root elongation of 35S-ABF2 plants. We did

not observe differences in the ABA inhibition of germination

(i.e. radicle emergence) between wild type and 35S-ABF2

plants (data not shown). However, altered ABA sensitivity

was observed during post-germination growth. Figure 4(a)

shows that growth of wild type (Ler) seedlings was inhibited

significantly in the presence of inhibitory amount of ABA (i.e.

1 lM ABA), and, therefore, only 13% of their cotyledons

expanded and turned green. In contrast, 76 or 90% of the

transgenic cotyledons expanded and turned green at the

same dose of ABA, demonstrating that aerial part growth of

35S-ABF2 seedlings was less sensitive to ABA. Inhibition of

root growth by ABA was also affected by ABF2 overexpres-

sion. Figure 4(b) shows that wild type root growth measured

by primary root elongation was inhibited gradually with

increasing amounts of ABA, reaching 25% of the control rate

on ABA-free medium at 10 lM ABA. 35S-ABF2 transgenic

root elongation was also inhibited by ABA, but with higher

degrees at all ABA concentrations tested. The result dem-

onstrates that primary root elongation of 35S-ABF2 plants

was hypersensitive to ABA. Taken together, ABF2 overex-

pression either enhanced or reduced, depending on tissue

type, ABA sensitivity during post-germination growth.

(b)

Time (h)18 20 22 24 26 28 30 32 34 36

Ger

min

atio

n (%

)

0

20

40

60

80

100

Ler

A204

A208

(c)

Ler A204 A208

Ler A204 A208

(d)(e)

Ler A204A208

Ler A204

A208

+sucrose–sucrose

A208

A204(a)

Ler

Figure 2. Growth phenotypes of 35S-ABF2 plants.

(a) Expression levels of ABF2 in wild type (Ler) and transgenic lines (A204 and A208) determined by RNA gel blot analysis. The bottom panel shows the ethidium

bromide-stained gel as a loading control.

(b) Germination assay. Seeds were plated on MS medium after 5 days of cold treatment at 4�C, and germination (fully emerged radicle) was scored (triplicates,

n ¼ 36 each) at various time points. The small bars represent standard errors.

(c) Three-week-old plants grown on soil.

(d) Six-week-old plants.

(e) Sucrose effect on seedling growth. Seeds were germinated and grown on MS medium with or without 1% sucrose for 10 days.

78 Sunmi Kim et al.


ABF2 overexpression promotes glucose-induced

developmental arrest process

High concentrations of sugars inhibit cotyledon greening/

expansion and true leaf formation of young seedling (Jang

et al., 1997). Many genetic studies showed that ABA is

essential for the developmental arrest process and that ABA

signaling components, ABI4 and ABI5, are integral compo-

nents of the glucose signal transduction (reviewed in

Gazzarrini and McCourt, 2001; Leon and Sheen, 2003). To

examine whether ABF2 is involved in the ABA-dependent

process, we determined the glucose sensitivity of 35S-ABF2

plants. When seeds were germinated and grown on a

medium supplemented with 3% glucose, which mildly

inhibits shoot development, the cotyledon greening effi-

ciency of wild type seedlings decreased to 72% (Figure 4c).

By contrast, only 30% (A204) or 35% (A208) of transgenic

cotyledons turned green at the same glucose concentration.

The increase in glucose inhibition of shoot development was

also observed at 4% glucose (Figure 4c), again demonstra-

ting the glucose hypersensitivity of 35S-ABF2 plants. The

hypersensitivity was not osmotic, i.e. it was not observed

with mannitol (data not shown).

Drought resistance of 35S-ABF2 plants

To investigate the possible role of ABF2 in water stress

response, whole plant survival rates of 35S-ABF2 plants

under water-deficit conditions were determined. When

6-day-old seedlings were withheld from water for

0 4 8 12 4 8 12

H2O (h) ABA (h)(b)

A204Ler A208

(a)

ABF2

rd29A

CHS

SUS1

Actin

A204 A208

ADH

A204 A208A204 A208 A204 A208Untreated Salt

Ler Ler

(c) +sucrose –sucrose

HXK2

HXK1

LerA20

4A20

8Ler A20

4A20

8

Actin

rbcS

Figure 3. Expression of ABA-regulated genes in 35S-ABF2 plants.

(a) RNA levels of ABA/stress-regulated genes were determined by RT-PCR

using total RNA isolated from 2-week-old seedlings. For salt treatment, roots

of plants were submerged in 250 mM NaCl for 4 h with gentle shaking before

harvest. The number of PCR cycles is different in untreated and salt-treated

samples.

(b) rbcS expression was determined by RNA gel blot analysis using 15 lg of

total RNA isolated from 3-week-old plants. Left, time course of ABA-

repression of rbcS expression. Plants were treated with 100 lM ABA or water

for indicated times before RNA isolation. Right, expression levels of rbcS in

35S-ABF2 plants. Bottom panels show ethidium bromide-stained gels.

(c) Expression levels of hexokinases, HXK1 and HXK2, were determined by

RT-PCR. RNA was isolated from germinating seedlings (3 or 5 days after

plating of wild type and transgenic seeds respectively) on MS media with or

without 1% sucrose.

1 µM ABA

Gre

en c

otyl

edon

s (%

)

0

20

40

60

80

100

(a)AK313AK405

Col-0

4%3%Glucose

Gre

en c

otyl

edon

s (%

)

40

20

0

60

80

100(c)

AK313AK405

Col-0

Rel

ativ

e ro

ot g

row

th (

%)

ABA (µM)0 0.5 1 5 10

0

20

40

60

80

100

(b)

AK313AK405

Col-0

Figure 4. ABA and glucose sensitivities of 35S-ABF2 plants.

(a) ABA effect on shoot development. Seeds were germinated and grown on

MS medium containing 1 lM ABA for 2 weeks, and seedlings with green

cotyledons were counted (triplicates, n ¼ 50 each).

(b) ABA effect on primary root elongation. Seeds were germinated for 3 days

on MS medium and the seedlings were transferred (n ¼ 6, triplicates) to fresh

medium containing various concentrations of ABA. Primary root length was

measured 7 days after the transfer, and relative growth compared with that on

ABA-free medium was indicated.

(c) Glucose effect on cotyledon greening. Seeds were germinated on MS

medium containing 3 or 4% glucose for 5 days, and seedlings with green

cotyledons were counted (quadruplicates, n ¼ 30 each). The small bars in (a),

(b), and (c) represent standard errors.



2 weeks, only 10 or 15% of the 35S-ABF2 plants survived

the treatment, whereas the wild type survival rate was

68% (Figure 5a). Thus, 35S-ABF2 seedlings were more

susceptible to water stress. When 3-week-old plants were

subjected to similar treatment, 97 or 100% of the 35S-

ABF2 plants survived, whereas only 22% of wild type

plants survived (Figure 5a). The result indicates that, un-

like the younger seedlings, 3-week-old 35S-ABF2 plants

were resistant to water stress. Additionally, transpiration

rates of the transgenic plants measured by fresh weight

loss of detached leaves were approximately 40% of the

wild type rate at this later growth stage (Figure 5b), sug-

gesting that ABF2 overexpression promoted stomatal

closure. Taken together, our results indicate that ABF2

overexpression enhanced or reduced water stress

resistance depending on the developmental stage when

the plants were subjected to water stress.

Enhanced tolerance of 35S-ABF2 plants to other abiotic

stresses

As ABA mediates adaptive responses not only to drought

but also to high salinity, extreme temperatures, and oxi-

dative stress, we investigated whether ABF2 overexpres-

sion altered the responses to these stresses. We first

determined the salt sensitivity of 35S-ABF2 plants. Salts

inhibit germination and seedling growth severely at high

concentrations and ABA is essential for normal response

to high salinity (Xiong and Zhu, 2002). When wild

type seeds were germinated and grown in a medium

(c)A204

A208A204

A208Ler Ler

MS 50 mM NaCl

(d)

A208(99%)

A204(95%)

Ler(10%)

(e)

MS

MV

38% 86%85%

A204 A208Ler

Surv

ival

rat

e (

%)

Youngstage

Oldstage

100

60

40

20

0

80

(a) LerA204A208

LerA204A208

LerA204A208

Time (min)L

eaf

wei

ght (

%)

0 40 80 120 160 200

100

60

40

20

0

80

(b)

10

30

50

70

90

Figure 5. Stress tolerance of 35S-ABF2 plants.

(a) Dehydration tolerance. Plants were withheld from water for 2 weeks, starting 6 days (young stage) or 3 weeks (old stage) after seed sowing, and then survival

rates were determined by counting plants that kept growing when re-watered. Each data point represents the mean of six determinations (n ¼ 30 each). 35S-ABF2

seeds were sown 3 days earlier than wild type seeds so that plants were at the similar developmental stages when the water-deficit condition was imposed.

(b) Transpiration rates. Rosette leaves (third and fourth true leaves) at similar developmental stages were detached and weighed at various times (triplicates, n ¼ 9

each). The small bars indicate standard errors.

(c) Salt tolerance. Seeds were germinated and grown on MS medium containing 50 mM NaCl for 10 days before the photograph was taken.

(d) Heat tolerance. Two-week-old (35S-ABF2) or 11-day-old (wild type) seedlings were exposed to 46�C for 1 h, and the photograph was taken after 7 days of recovery

period at normal growth temperature. Survival rates, determined by counting the number of plants that kept growing after the treatment, are shown in parentheses

(triplicates, n ¼ 20 each).

(e) Oxidative stress tolerance. Rosette leaves (third and fourth true leaves) from 3-week-old (wild type) or 24-day-old (35S-ABF2) plants grown on soil were floated in

6 lM MV for 43 h before the photograph was taken. Chlorophyll contents relative to those of the mock-treated samples are shown in parentheses (triplicates, n ¼ 16

each).

80 Sunmi Kim et al.


containing 50 mM NaCl, primary root elongation and lat-

eral root initiation or growth were severely inhibited

(Figure 5c). Primary root growth of 35S-ABF2 seedlings

was similarly affected, but their lateral root growth was

comparable with that in the salt-free medium, indicating

that lateral root growth of the transgenic plants is less

sensitive to salt. Reduced sensitivity was not observed

with mannitol (data not shown), suggesting that it is salt-

specific rather than osmotic.

Next we investigated the heat tolerance of 35S-ABF2

plants. Plants at the same developmental stage were

exposed to lethal temperature conditions (i.e. 46–48�C under

our experimental conditions), and then whole plant survival

rates were determined after a recovery period. When 11-day-

old wild type plants were exposed to 46�C for 1 h, 10% of

them survived (Figure 5d). In contrast, 95 or 99% of the 35S-

ABF2 plants at the same developmental stage (i.e. 14-day

old) survived the same temperature condition, indicating

that 35S-ABF2 plants are heat-tolerant.

Finally, we determined the oxidative stress tolerance of

35S-ABF2 plants by evaluating damage induced by the

chemical methylviologen (MV). MV, an herbicide (i.e. para-

quat), causes chlorophyll degradation and the leakage of cell

membrane by generating reactive oxygen species (Kurepa

et al., 1998; Slade, 1966). Rosette leaves at the same

developmental stage were placed in a 6 lM MV solution.

The damage induced by the treatment was then inspected

visually. Wild type leaves became bleached with longer

incubations, and quantitative determination of their chloro-

phyll contents indicated that they lost 62% of their chloro-

phyll contents after 43 h compared with the mock-treated

control leaves (Figure 6e). By contrast, transgenic leaves

remained green at the same condition and lost only 14–15%

of their chlorophyll contents. Thus, 35S-ABF2 transgenic

leaves were less susceptible to the damage induced by MV.

We also tested the effect of MV on seedling growth, by

germinating and growing them on a medium containing

MV, and observed the enhanced tolerance of 35S-ABF2

plants (data not shown).

An abf2 null mutant is defective in glucose response and

grows faster

The function of ABF2 was further investigated by analyzing

its mutant phenotypes. A mutant line, in which a T-DNA is

inserted in the first intron, was obtained from the Arabid-

opsis stock center, and, after confirming the T-DNA insertion

and the abolishment of ABF2 expression (Figure 6a), various

phenotypes were scored.

The abf2 null mutant plants germinated normally (data

not shown), but the mutant seedlings were larger than wild

type plants during vegetative growth phase (Figure 6b). The

size difference was most evident in the middle of seedling

growth phase (i.e. with 15-day-old seedlings) and dimin-

ished afterwards. At the bolting stage, abf2 plants were of

the same size as the wild type plants. Thus, abf2 mutation

promoted growth during the early phase of seedling growth.

Together with the retarded growth of 35S-ABF2 seedlings,

the result indicated that ABF2 is involved in seedling growth

regulation under normal conditions.

The abf2 mutant plants also displayed reduced sensitivity

to glucose. For instance, addition of 3% glucose to germi-

nation medium lowered the cotyledon greening efficiency of

wild type (Col-0) seedlings to 62% (Figure 6c). In contrast,

cotyledon greening was observed in 75% of transgenic

seedlings on the same medium. At 4% glucose, 19% of the

wild type (Col-0) cotyledons expanded and turned green,

whereas 53% of the abf2 mutant cotyledons did. The results

demonstrate that ABF2 is a component necessary for the

glucose-triggered developmental arrest process.

The abf3 and abf4 mutants are defective in ABA and stress

responses

We did not observe significant phenotypic changes in ABA or

stress response with the abf2 mutant (data not shown), ex-

cept faster seedling growth and partial glucose insensitivity.

Overexpression of ABF3 and ABF4 inhibits germination or

seedling growth and enhances ABA, salt, and glucose sen-

sitivities together with drought tolerance (Kang et al., 2002).

Some of these phenotypes are similar to those of ABF2

Gre

en c

otyl

edon

s (%

)

4%3%Glucose

40

20

0

60

80

100(c)AK218

Col-0

Actin

ABF2

(a)

Col-0

AK218

Rel

ativ

e w

eigh

t (%

)

020406080

100120140

Time (day)10 15 20 25

(b)

Figure 6. Phenotypes of an abf2 knockout mutant.

(a) ABF2 expression levels in seedlings were determined by RT-PCR. AK218

denotes the mutant line.

(b) Growth of the abf2 mutant plants. Aerial parts of seedlings grown on soil

were weighed at various time points and relative weights compared with

those of wild type (Col-0) plants are indicated. Each data point represents the

mean of eight determinations (n ¼ 6 each).

(c) Glucose sensitivity of the mutant plants. Seeds were germinated on MS

medium containing 3 or 4% glucose for 5 days, and seedlings with green

cotyledons were counted (quadruplicates, n ¼ 30 each). Standard errors are

indicated by the small bars.



overexpression. The lack of the abf2mutant phenotypes thus

may be due to the possible functional redundancy among

ABF family members. To test the hypothesis, we obtained

abf3 and abf4 mutants and investigated their phenotypes.

One abf3 and one abf4 knockout mutant lines were

identified among a number of T-DNA insertion lines avail-

able from the Arabidopsis stock center (Figure 7a). As

shown in Figure 7(b), germination assay revealed that the

abf4 null mutant seeds germinated more efficiently than

wild type seeds, the time taken for 50% germination of the

mutant seeds being 5 h less than that for wild type seeds.

Similarly, the abf3 mutant seeds germinated faster than wild

type (Figure 7b), although their germination rate was

slightly slower compared with that of the abf4 mutant. The

mutants were defective in ABA response as well. ABA dose–

response analysis indicated that the germination of the abf3

(g)

Col-0

AK313

AK405

Time (h)18 20 22 24 26 28 30 32

Ger

min

atio

n (%

)

0

20

40

60

80

100(b)

Col-0AK313AK405

40 80 120 160 2000Time (min)

Lea

f w

eigh

t (%

)

0

10

20

30

40

50

60

70

80

90

100(h)

Ger

min

atio

n (%

)

0

20

40

60

80

100

0 0.5 1 2 5ABA (µM)

(c)

AK313AK405

Col-0

0 0.5 1 2 5ABA (µM)

Roo

t elo

ngat

ion

(%)

0

20

40

60

80

100

(d)AK313AK405

Col-0

Salt (mM)0 50 100 150

Ger

min

atio

n (%

)

20

40

60

80

100

0

(e)

AK313AK405

Col-0

Salt (mM)0 50 100 150 200

Roo

t elo

ngat

ion

(%)

0

20

40

60

80

100(f)

AK313AK405

Col-0

ABF3Col-

0AK31

3

ABF4Col-

0AK40

5(a)

Actin Actin

Col-0 AK313

Col-0 AK405

(42.6%) (0%)

(60%) (19.9%)

Figure 7. Phenotypes of abf3 and abf4 knockout

mutants.

(a) Expression levels of ABF3 and ABF4 in their

mutant lines (AK313 and AK405 respectively)

were determined by RT-PCR.

(b) Germination assay. Seeds were plated on MS

medium after 5 days of cold treatment at 4�C,

and germination (radicle emergence) was scored

at various time points (triplicates, n ¼ 36 each).

(c) ABA dose–response analysis of germination.

Germination assay was carried out as in (b) on a

medium containing various amounts of ABA.

Germination was scored 4 days after plating

(triplicates, n ¼ 36 each).

(d) ABA dose–response analysis of primary root

elongation. Seeds were germinated on MS

medium for 3 days, transferred to media con-

taining various amounts of ABA, and root length

was measured 5 days after the transfer. Relative

elongation compared with that on ABA-free

control medium is shown (triplicates, n ¼ 6

each).

(e), (f) Salt sensitivity. Germination and root

elongation assays were performed on media

containing various concentrations of NaCl. For

root growth, relative elongation compared with

that on salt-free control medium is shown.

(g) Drought resistance. One-week-old seedlings

were withheld from water for 2 weeks, and the

photograph was taken after 2 days of recovery

period. The numbers indicate survival rates

(quadruplicates, n ¼ 30 each).

(h) Transpiration rates. Rosette leaves (third and

fourth true leaves) from 3-week-old plants were

detached and weighed at various time points

(triplicates, n ¼ 10 each). The small bars indicate

standard errors.

82 Sunmi Kim et al.


mutant seeds was less sensitive to ABA inhibition than wild

type seeds at medium concentrations (i.e. 1 and 2 lM ABA)

(Figure 7c). Reduced ABA sensitivity during germination

was also observed with the abf4 mutant at 1 lM ABA

(Figure 7c). Likewise, similar ABA dose–response analysis

showed that primary root elongation of the abf3 and the

abf4 mutant seedlings was partially insensitive to ABA

(Figure 7d).

Furthermore, the abf3 and the abf4 mutants exhibited

abnormal responses to high salt and water stress.

Figure 7(e) shows that germination of wild type seeds was

inhibited gradually with increasing amounts of NaCl. In

comparison, germination of the mutant seeds, especially

that of abf3, was less sensitive to salt inhibition. In the case

of abf4 mutant, primary root elongation was also insensitive

to salt, although the degree of insensitivity was small

(Figure 7f). We also examined the dehydration tolerance of

the mutant plants, by determining their survival rates under

water-deficit conditions. Figure 7(g) shows that the survival

rates of the mutant seedlings were substantially lower than

that of wild type plants, indicating that they were more

susceptible to water stress. Moreover, transpiration rate of

the abf3 mutant determined by fresh weight loss of detached

leaves was slightly higher than the wild type rate (Figure 7h),

suggesting that the abf3 mutation probably resulted in the

partial impairment of stomatal closing. Compared with wild

type plants, the abf3 and the abf4 mutant plants thus

germinated more efficiently, displayed reduced sensitivities

to ABA and salt, and were susceptible to water stress.

The phenotypes indicate that both ABF3 and ABF4 are

required for normal ABA/stress responses and that they play

more important roles in ABA/stress responses than ABF2.

However, the abf3 and the abf4 mutations had little effects

on glucose response (data not shown).

Discussion

Our data suggest that ABF2 is involved in ABA and stress

responses. Its overexpression altered the expression of ABA/

stress-regulated genes and conferred several ABA-associ-

ated phenotypes that include growth retardation, altered

ABA and glucose sensitivities, and enhanced tolerance

to several abiotic stresses. Its mutant phenotypes indicate

its function in seedling growth regulation and glucose

response.

ABF2 is a positive component of glucose signal transduction

Abscisic acid and sugars have similar effects on seedling

growth. Both promote growth at low doses, but inhibit it at

high concentrations. In addition, ABA and sugars both sup-

press the expression of photosynthetic genes (Jang and

Sheen, 1994; Weatherwax et al., 1996). Recent genetic stud-

ies show that all ABA-deficient mutants and some ABA

signaling mutants (i.e. abi4 and abi5) are insensitive to

sugars, indicating that ABA and part of its signaling cascades

are integral components of the sugar response pathway

(reviewed in Gazzarrini and McCourt, 2001; Leon and Sheen,

2003). The glucose hypersensitivity of 35S-ABF2 plants

(Figure 4c) and the glucose insensitivity of the abf2 mutant

seedlings (Figure 6c) demonstrate that ABF2 is also a positive

component of the glucose signal transduction.

ABF2 is involved in seedling growth regulation

Retarded growth/maturation of 35S-ABF2 plants and faster

growth of its mutant seedlings indicate that ABF2 is involved

in the process of seedling growth regulation. Consistent

with this, ABF2 promoter is very active in the emerging

shoots of germinating seedlings (Figure 1b, panel c).

Moreover, we observed slightly (approximately 2 days)

earlier flowering of the mutant plants under water-deficit

conditions (data not shown). At present, we do not know

how ABF2 affects seedling growth. However, several

observations raised the possibility that it may affect growth

by regulating genes associated with energy supply or util-

ization. First, growth retardation of 35S-ABF2 seedlings was

alleviated by supplementing growth medium with sucrose

(Figure 2d), implying that this may result from a deficiency

in energy source. Secondly, ABF2 overexpression sup-

pressed the rbcS expression. rbcS is the key enzyme to

determine photosynthetic rate, catalyzing the first step of

carbon fixation. As rbcS is one of the most abundant pro-

teins, the decrease in the level of rbcS observed in ABF2

transgenic lines may have little effect on the growth of plants

with full photosynthetic capacity. However, it may impair the

photosynthetic capacity of germinating seedlings that are in

transition to the autotropic state, thus slowing down their

growth. Thirdly, ABF2 abolished the expression of the genes

encoding hexokinases (HXK1 and HXK2) (Figure 3c), which,

as sugar sensors (Jang et al., 1997), catalyze the first reac-

tion of glycolysis. Their deficiency, therefore, may have

caused limitation in energy utilization. This hypothesis is

supported by the fact that recovery of seedling growth by

exogenous sucrose was accompanied by recovery of their

expression to wild type levels (Figure 3c). One of the ways

plants survive abiotic stresses is metabolic arrest (i.e. stop of

growth and development) (Pastori and Foyer, 2002), which is

also caused by high levels of ABA. Together with the stress

and ABA inducibility of ABF2 expression, these observations

imply that ABF2 might be involved in the metabolic arrest

process by regulating genes involved in energy metabolism.

The ABF2 activity may be modified by developmental and

environmental factors

The transcriptional regulatory function of ABF2 was sup-

ported by the altered expression of a number of ABA- or



sugar-regulated genes in 35S-ABF2 plants (Figure 3). Fur-

thermore, our data suggest that ABF2 activity may be

affected by several factors. First, it played a negative regu-

latory role for several genes under normal conditions but a

positive role for the same genes under high salt conditions

(Figure 3a), implying that high salinity-induced post-trans-

lation modification was necessary for its gene activation

function. Secondly, the downregulation of HXK1 and HXK2

in a medium lacking sucrose, but their normal expression in

a medium containing sucrose (Figure 3c), suggests that

ABF2 activity as well as its expression may be modified by

sucrose. Thirdly, the developmental stage dependence of

HXK repression and several phenotypes including ABA

sensitivity (Figure 4a,b) and drought tolerance (Figure 5a)

implies that its activity may also be subject to developmental

regulation. The nature of these putative modifications is not

known at present. It may be phosphorylation or dephosph-

orylation, as has been demonstrated for ABA activation of its

(i.e. AREB1) activity in protoplasts (Uno et al., 2000). Con-

sidering the diverse roles of ABA and the multiplicity of its

signaling pathways (Finkelstein et al., 2002), it can be spe-

culated that the modification may involve interaction(s) with

other transcription regulatory or signaling components. In

fact, our preliminary analysis of its interacting partners

suggests such a possibility (data not shown).

ABF2 overexpression affects multiple stress tolerance

Although there are ABA-independent stress response path-

ways and, thus, not all stress responses are mediated by

ABA (Shinozaki and Yamaguchi-Shinozaki, 2000), ABA is

essential for the full protection against most of the common

abiotic stresses: drought, freezing, high salinity, high

temperature, and oxidative stress (Larkindale and Knight,

2002; Schroeder et al., 2001; Thomashow, 1999; Xiong et al.,

2002). Our results indicate that ABF2 is likely to be involved

in the adaptive processes to these common abiotic stresses.

Its overexpression affected the whole plant survival rate

under water-deficit conditions (Figure 5a). The reduced

transpiration rates of 35S-ABF2 plants (Figure 5b) and its

strong promoter activity in guard cells (Figure 1b, panel f)

suggest its active role in stomatal closure. Moreover,

enhanced tolerance to high salt, heat, and oxidative stress

suggests that ABF2 probably plays a role in the adaptation to

these stresses as well.

Specificity and redundancy of the functions of ABF family

members

The ABF subfamily of bZIP factors consists of four members

(Choi et al., 2000), and there is another subfamily consisting

of embryo-abundant, four ABF-related factors that include

ABI5 (Bensmihen et al., 2002; Kim et al., 2002). Hence,

functional redundancy among the ABF family members and,

possibly, those of ABI5/AtDPBF subfamily is anticipated. In

fact, ABF2, ABF3, ABF4, and ABI5 overexpression lines dis-

play common phenotypes such as ABA hypersensitivity of

root growth, lower transpiration rate, and glucose hyper-

sensitivity. In addition, ABF3 and ABF4 overexpression lines

are resistant to multiple stresses (data not shown, see

Table 1).

However, a number of observations indicate that ABF

family members play nonetheless distinct roles in ABA/

stress responses. They differ from each other in both

stress-induction patterns and their transcriptional activity

levels (Choi et al., 2000), suggesting that each of them

Table 1 Overexpression phenotypes of ABFs

ABFs Affected genes

Phenotypes

Growth ABA/glucose responsesa Stress tolerance

ABF1b Not determined Faster growth,early flowering

Not observed Not observed

ABF2 rbcS, HXK1, HXK2, SUS1, CHS, ADH1 Slow germination,slow growth

ABA: ›fl glucose: › Droughtc, salt, heat, oxidative

ABF3d Rd29B, rab18, ICK1, ABI1, ABI2, KAT1,KAT2, ADH1, CHS

Slow germination,minor dwarfism

ABA: › glucose: › Drought, (salt)e, chilling, freezing,heat, oxidative

ABF4d Rd29B, rab18, ICK1, ABI1, KAT2, SKOR,ADH1, CHS

Severe dwarfism ABA: › glucose: › Drought, (salt)e, freezing, heat,oxidative

ABF2-specific phenotypes and the genes affected by ABF2 only are underlined, phenotypes complementary to knockout mutant phenotypes areindicated in boldface, and stress tolerance not shown in this paper are in italics.a›, hypersensitive; fl, insensitive; ›fl, hypersensitive or insensitive depending on tissue type.bData not shown.cSusceptible or tolerant depending on developmental stage.dKang et al. (2002).eSalt-hypersensitive at the germination stage.

84 Sunmi Kim et al.


functions under specific conditions and that their gene

activation capacity or mechanisms differ from each other.

The latter hypothesis is supported by observations (Kang

et al., 2002) that different subsets of ABA/stress-responsive

genes are affected by overexpression (Table 1). For

instance, downregulation of rbcS and HXKs was observed

only in 35S-ABF2 plants, whereas expression of several

ABF3- or ABF4-regulated genes (e.g. rab18, ICK1, ABI1,

SKOR, etc.) was not affected in 35S-ABF2 plants. Besides,

their overexpression phenotypes were distinct in several

respects (Table 1). ABA insensitivity of shoot growth

(Figure 4a), salt tolerance (Figure 5c), and drought suscep-

tibility of young seedlings (Figure 5a) were unique to 35S-

ABF2 plants. On the contrary, 35S-ABF3 and 35S-ABF4

seedlings were salt-hypersensitive (Kang et al., 2002), and,

in contrast to 35S-ABF2 plants, their responses to ABA and

dehydration did not exhibit tissue or developmental stage

dependence. With ABF1, we did not observe ABA- or

stress-related phenotypes except slightly faster growth

(data not shown).

The knockout mutant phenotypes further revealed the

differential functions of ABF family members. Although the

abf2 mutant was normal in germination and ABA/stress

responses, abf3 and abf4 mutants germinated more effi-

ciently than wild type plants and were defective in ABA/

stress responses. These observations demonstrate that

ABF3 and ABF4 are required for normal germination and

ABA/stress responses, whereas ABF2 is not. However, ABF2

is necessary for the regulation of post-germination growth

in the presence of glucose and even under normal condi-

tions, as mentioned above. In conclusion, our data indicate

that ABF2, ABF3, and ABF4 are involved in ABA, stress, and

glucose responses to various degrees and that, whereas

ABF3 and ABF4 play essential roles in germination control

and ABA/stress responses, ABF2 plays a more important

role in seedling growth regulation and glucose response.

Experimental procedures

Plant growth

Arabidopsis thaliana, ecotype Landsberg erecta (Ler) and Columbia(Col-0), were used. Plants were grown on soil (1:1:1 mixture of peatmoss, vermiculite, and perlite) or aseptically on Murashige–Skoog(MS) medium supplemented with 0.8% agar, at 22�C under long dayconditions (16 h light/8 h dark cycles). Unless stated otherwise, theMS medium was supplemented with 1% sucrose, and the plants onsoil were watered once a week by the partial immersion of pots inwater containing 0.1% Hyponex (Hyponex Co., Marysville, OH, USA).

RNA isolation and RNA gel blot analysis

RNA isolation, RNA gel blot analysis and coupled RT-PCR wereperformed as described (Kang et al., 2002). The abscence of con-taminating DNA in RNA preparations used in RT-PCR was confirmed

first by using the actin primer set spanning an intron and further byusing primer sets spanning introns whenever possible. The RT-PCRresults were confirmed by several independent reactions. Someprimer sets used in the RT-PCR of ABA responsive genes were des-cribed previously (Kang et al., 2002). For hexokinase genes, primersets 5¢-gtaaagtagctgttggagcgac-3¢ and 5¢-cattctgtagcgacaaacttcgc-3¢,and 5¢-gtaaagtggcagttgca acgacg-3¢ and 5¢-cgccttctgtagcgacaaacttg-3¢ were used for HXK1 and HXK2 respectively. 5¢-gctagtggtgtggttc-cagttc-3¢ and 5¢-ctgagctcttgcagcaacctg-3¢ primers were used for theRT-PCR of ABF2, and an ABF2-specific probe (N-terminal 274 bpfragment) was used in its RNA gel blot analysis. For the RNA gel blotof rbcS, the coding region of ats1B (accession no. X14564) was usedas a hybridization probe.

Generation of transgenic plants

To generate 35S-ABF2 plants, the coding region of ABF2 includingthe stop codon was amplified by PCR using Pfu polymerase. For theeasiness of cloning, BamHI and SacI linker sequences were attachedbefore the initiation and after the stop codons respectively. AfterBamHI and SacI double digestion, the fragment was ligated withpBI121 (Jefferson et al., 1987), which was prepared by excising theGUS coding region after digestion with the same restriction enzymeset. The intactness of the junction sequence was confirmed by DNAsequencing. Transformation of Arabidopsis was performed by thevacuum infiltration method (Bechtold and Pelletier, 1998), usingAgrobacterium tumefaciens strain GV3101. Fifteen T3 homozygouslines were recovered and, after preliminary analysis of the trans-genic lines, two representative lines with high ABF2 expressionlevels were chosen for detailed phenotypic analysis.

The ABF2 promoter-GUS construct was prepared by joining a 5¢flanking sequence (2.3 kb from the initiation codon) to the GUScoding region of PBI101.2. The promoter fragment was prepared byPCR, using the primer set 5¢-ccgcaggagatatgaggaattagaacc-3¢ and5-cctaataactggatctgaaccatgaatac-3¢, and ligated with pBI101.2digested with SmaI. Histochemical GUS staining was performedas described (Kang et al., 2002), using T1 or T2 generation plants.

Knockout mutants

Seeds of knockout mutant lines were obtained from the Arabidopsisstock center. Stock seeds were germinated and grown on soil, andseeds were harvested from individual plants. After testing the seg-regation of kanamycin resistancy (KanR), homozygous sublinesexhibiting 100% KanR were established from plants whose progenysegregate with 3:1 ratio of KanR and KanS seeds in the cases of ABF2and ABF3 knockout lines. DNA was isolated from the selected lines,and T-DNA insertion in the annotated positions was confirmed byPCR and subsequent sequencing of the amplified fragments beforephenotype analysis. In the case of the ABF4 knockout line, progenyof the most of the plants exhibited 100% KanR, and T-DNA insertionand knockout were confirmed in randomly chosen plants all ofwhose progeny showed KanR. Among the available lines, we wereable to identify one knockout line for each of ABF2 (SALK_002984),ABF3 (SALK_075836), and ABF4 (SALK_069523) respectively. T-DNAis inserted in these lines in the first intron (ABF2), in the leader intron(ABF3), or in the first exon (ABF4).

Phenotype analysis and stress tests

Germination (full emergence of radicles) was scored on MS med-ium containing 1% sucrose. For the ABA dose–response analysis of



germination, sucrose-free MS medium was used. Seeds collected atthe same time were used. Root elongation assay, water stress testsand transpiration rate measurements were carried out as described(Kang et al., 2002).

For oxidative stress tests, third and fourth true rosette leavesfreshly detached from plants at similar development stages werefloated abaxial side up on a methyl viologen (Sigma M2254,St Louis, MO, USA) solution in MS in the light for the specifiedtime. Chlorophyll contents of the detached leaves were measuredaccording to Lichtenthaler (1987) at the end of the treatments. Heattreatments for survival rate determination were performed byexposing, without any pre-treatments, plants on Petri plates to46–48�C for specified times, and photographs were taken after7 days of recovery period at 22�C. Plates were kept sealed during theheat treatment and the recovery period.

Acknowledgements

We are grateful to Dr Young-Min Woo and Dr Moon Soo Soh forcritical reading of the manuscript. This work was supported inpart by grants from the Biogreen 21 program funded by RDA ofKorea, the Agricultural Plant Stress Research Center of the SRCprogram funded by KOSEF, and the Crop Functional GenomicsCenter of the 21C Frontier Program funded by MOST to S. Y. Kim.This paper is Kumho Life and Environmental Science Laboratorypublication no. 70.

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ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance