Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene...

Impaired sucrose-induction mutants reveal the modulationof sugar-induced starch biosynthetic gene expression byabscisic acid signalling

Fred Rook, Fiona Corke, Roderick Card, Georg Munz, Caroline Smith and Michael W. Bevan*

Department of Molecular Genetics, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Received 3 October 2000; revised 20 February 2001; accepted 6 March 2001.*For correspondence (fax +44 1603 450025; e-mail [email protected]).

Summary

Plants both produce and utilize carbohydrates and have developed mechanisms to regulate their sugar

status and co-ordinate carbohydrate partitioning. High sugar levels result in a feedback inhibition of

photosynthesis and an induction of storage processes. We used a genetic approach to isolate components

of the signalling pathway regulating the induction of starch biosynthesis. The regulatory sequences of the

sugar inducible ADP-glucose pyrophosphorylase subunit ApL3 were fused to a negative selection marker.

Of the four impaired sucrose induction (isi) mutants described here, two (isi1 and isi2) were speci®c to this

screen. The other two mutants (isi3 and isi4) showed additional phenotypes associated with sugar-

sensing screens that select for seedling establishment on high-sugar media. The isi3 and isi4 mutants

were found to be involved in the abscisic acid signalling pathway. isi3 is allelic to abscisic acid insensitive4

(abi4), a gene encoding an Apetala2-type transcription factor; isi4 was found to be allelic to glucose

insensitive1 (gin1) previously reported to reveal cross-talk between ethylene and glucose signalling. Here

we present an alternative interpretation of gin1 as an allele of the ABA-de®cient mutant aba2. Expression

analysis showed that ABA is unable to induce ApL3 gene expression by itself, but greatly enhances ApL3

induction by sugar. Our data suggest a major role for ABA in relation to sugar-signalling pathways, in that

it enhances the ability of tissues to respond to subsequent sugar signals.

Keywords: Arabidopsis, sugar signalling, starch, abscisic acid, isi, AGPase.

Introduction

Plants produce sugars during photosynthesis in green

tissues and transport them for subsequent metabolism or

storage in heterotrophic tissues. The disaccharide sucrose

is the main exported sugar, and this and other assimilates

are partitioned for different uses in response to physio-

logical, developmental and environmental cues. In add-

ition to its role as the major transported assimilate,

sucrose is an important mobile signal molecule in plants

(Smeekens, 2000; Smeekens and Rook, 1997). Sugar-

mediated signals are integrated with a wide range of

other signalling pathways, such as those regulating

responses to N availability, light conditions, growth regu-

lators and stress, to permit a balanced distribution of

carbon moieties (Koch, 1996; Smeekens, 2000). This par-

titioning of carbohydrates is achieved in part by distinct

patterns of gene expression in source and sink tissues

(Herbers and Sonnewald, 1998; Koch, 1996).

In photosynthetic tissues, the expression of genes

encoding components of photosynthetic complexes is

repressed by high sugar levels (Krapp et al., 1991, Krapp

et al., 1993). High sugar levels also lead to the reciprocal

induction of genes associated with sink metabolism, such

as those encoding enzymes of starch biosynthesis. How

plants determine their carbohydrate status (`sugar sens-

ing'), and how this results in distinct responses for

different genes and tissues, is still largely unknown. A

role for hexokinase as a sugar sensor has been proposed,

but other sugar-sensing mechanisms are thought to exist

(Jang et al., 1997; Rook et al., 1998; Smeekens and Rook,

1997). Plant proteins similar to the yeast protein kinase

SNF1, required for de-repression of glucose-repressed

gene expression in yeast, have been identi®ed and impli-

cated in sugar-mediated gene expression in plants

(Halford and Hardie, 1998; Purcell et al., 1998). The use of

The Plant Journal (2001) 26(4), 421±433

ã 2001 Blackwell Science Ltd 421

genetic approaches has resulted in the isolation of sugar-

response mutants (Dijkwel et al., 1997; Martin et al., 1997;

Mita et al., 1997a; Mita et al., 1997b; Zhou et al., 1998), and

some of the corresponding genes have been identi®ed.

The prl1 mutation, conferring hypersensitivity to glucose

and sucrose, encodes a nuclear WD protein (NeÂmeth et al.,

1998) and was found to interact with Arabidopsis SNF1-like

protein kinases (Bhalerao et al., 1999). Recently it was

reported that the sun6 mutant, showing reduced carbohy-

drate repression of photogene expression during seedling

development, encodes the AP2-domain transcription fac-

tor ABSCISIC ACID INSENSITIVE4 (ABI4; Huijser et al.,

2000). A role for abscisic acid (ABA) signalling in sugar

regulation of plant vegetative development was implied by

the observation that ABA-de®cient and several ABA-

insensitive mutants are insensitive to high levels of

glucose and sucrose (Arenas-Huertero et al., 2000; Huijser

et al., 2000; Laby et al., 2000).

The transcription of several genes involved in starch

biosynthesis is induced by sugars (Koûmann et al., 1991),

including genes encoding ADP-glucose pyrophosphory-

lase (E.C. 2.7.7.27; AGPase) subunits. AGPase catalyses the

synthesis of ADP-glucose and pyrophosphate from glu-

cose-1-phosphate and ATP. ADP-glucose is used as the

glycosyl donor by starch synthases. It is thought that in

angiosperms AGPase is composed of two small and two

large subunits (Morell et al., 1987; Smith-White and Preiss,

1992). The allosteric regulation of AGPase activity is a

major physiological control mechanism for starch bio-

synthesis in plants (Stark et al., 1992), but AGPase tran-

script levels also contribute to starch production

(Sweetlove et al., 1999). The expression of genes encoding

AGPase subunits is differentially regulated by tissue

speci®c and physiological signals. Sugar-induced expres-

sion has been described for several AGPase subunits (Bae

and Liu, 1997; MuÈ ller-RoÈ ber et al., 1990; Sokolov et al.,

1998). In Arabidopsis, genes encoding one small (ApS) and

three large (ApL1, ApL2 and ApL3) subunits have been

identi®ed to date (Villand et al., 1993).

We have conducted a genetic analysis of the sugar-

regulated expression of the ApL3 gene using a fusion of

the ApL3 regulatory sequences with a negative selection

marker. A transgenic line containing this construct was

used to select for mutants in which the ApL3 promoter is

no longer induced by high sugar levels. In this report we

describe the isolation and characterization of four impaired

sucrose induction (isi) mutants. While isi1 and isi2 were

unique to this screen, isi3 and isi4 were found to be allelic

to previously identi®ed sugar-response mutants. isi3 was

identi®ed as ABI4 (Arenas-Huertero et al., 2000; Finkelstein

et al., 1998; Huijser et al., 2000; Laby et al., 2000), while isi4

was shown to be allelic to glucose insensitive1 (gin1; Zhou

et al., 1998). The isi4/gin1 mutants were found to display

ABA-de®cient phenotypes, and their proposed identity as

alleles of aba2 is discussed. Our results show that sugar

induction of starch biosynthetic genes, like carbohydrate

repression of seedling development and concomitant

induction of photosynthetic gene expression (Arenas-

Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000),

depends on ABA signalling. Arenas-Huertero et al. (2000)

recently suggested that glucose responses were mediated

through ABA signalling. We present an alternative model

in which ABA is not directly involved in sugar signalling,

but regulates the way in which tissues respond to a

separate sugar signal.

Results

The ApL3 gene encodes a sink tissue-speci®c AGPase

large subunit in Arabidopsis

Our genetic approach to isolating components of the

pathway regulating sugar induction of starch biosynthetic

genes used a fusion of the regulatory sequences of a

sugar-inducible AGPase subunit to a negative selection

marker. Several genes encoding AGPase small and large

subunits are present in angiosperms. In Arabidopsis, one

gene encoding a small subunit (ApS) and three genes

encoding large subunits (ApL1, ApL2 and ApL3) were

originally identi®ed by Villand et al. (1993). However, the

Arabidopsis Genome Initiative (AGI) has recently revealed

the existence of a second small subunit (designated

At1g05610 by the AGI) and an additional large subunit

(At2g21590). Expression of the ApL3 gene was highly

responsive to external sucrose (Figure 1a), and therefore

most suitable for identifying mutants affecting its sugar-

induced expression. A genomic fragment containing the

ApL3 gene was isolated by screening a BAC library (see

Experimental procedures) using an ApL3 cDNA fragment

(Villand et al., 1993) as a probe. The region encoding ApL3

was subcloned and sequenced (GenBank accession num-

ber Y18432; AGI designation At4g39210). The predicted

gene structure was con®rmed by sequencing a near full-

length cDNA. The ApL3 gene consists of 14 exons and,

with the exception of the N-terminal transit peptide, its

deduced amino-acid sequence is highly homologous with

that of other AGPase large subunits (typically 80% identity

for the mature protein).

The ApL3 protein-coding region was replaced at the ATG

start codon with either the GUS reporter gene (Jefferson

et al., 1987) or a bacterial cytochrome P450 gene (O'Keefe

et al., 1994) which served as a negative selection marker.

These constructs contained approximately 4.5 kb of the 5¢upstream regulatory region and 1.5 kb of the 3¢ region.

Transgenic Arabidopsis containing the GUS construct was

used for histochemical analysis of the ApL3 tissue-speci®c

expression. Five independent transgenic lines showed

identical GUS-staining patterns, and only differed in the

422 Fred Rook et al.

ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 421±433

level of GUS activity. GUS activity was observed in most

tissues with the exception of roots, consistent with RNA

gel-blot analysis of ApL3 expression (data not shown). The

results for line A3G5 are shown in Figure 2. In both

greenhouse-grown and sterile culture-grown plants, GUS

expression was associated with the vascular tissue in the

leaf blades and petioles (Figure 2a). Thin sections showed

that GUS expression was restricted to the starch sheath

layer below the major veins (Figure 2b). Staining with

Lugol's solution showed the presence of starch granules in

this cell layer, con®rming its identity as the starch sheath

(data not shown). When detached leaves were ¯oated on a

200 mM sucrose solution, GUS expression was inducible

in mesophyll cells (Figure 2a), showing that the regulatory

sequences necessary for sugar induction were present in

the construct. Additional GUS expression was observed in

elongating stamens and the pistil in ¯owers, and during

the early stages of seed development (Figure 2c). GUS

expression was no longer detectable after the heart stage

of embryo development, when seeds break down starch

and start to accumulate lipids.

The tissue-speci®c expression of the ApL3 gene is

similar to that reported for other strongly sugar-inducible

AGPase subunits such as AGPase-S and agpB1 of potato.

For these genes, expression was also observed in the

starch sheath layer of leaf veins and petioles (du Jardin

et al., 1997; MuÈ ller-RoÈ ber et al., 1994). Using amino-acid

sequence comparison, Smith-White and Preiss (1992)

divided AGPase large subunits into classes expressed in

photosynthetic and non-photosynthetic tissues. Park and

Chung (1998) previously placed ApL3 with the potato

AGPase-S and tomato AgpL1 in the subgroup with

expression in non-photosynthetic tissues. Both its

sequence homology and tissue-speci®c expression are

consistent with the characterization of ApL3 as a sink

tissue-speci®c AGPase subunit in Arabidopsis.

Isolation of impaired sucrose-induction mutants

To isolate mutants in the sugar-inducible expression of the

ApL3 gene, we fused its regulatory sequences to a

bacterial cytochrome P450 gene, P450su1, that metabolizes

the non-herbicidal sulfonylurea R7402 compound to a

highly phytotoxic form (O'Keefe et al., 1994). For its

activity, the P450su1 requires targeting to the chloroplast

where it can be reduced by ferredoxin. The plasmid pSSU-

SU121 (O'Keefe et al., 1994) contains a translational fusion

of the petunia Rubisco small subunit promoter and transit

peptide with P450su1. Using a PCR- and linker-based

strategy, we replaced the original promoter and polyade-

nylation signal sequences of the Rubisco small subunit

with those of the Arabidopsis ApL3 gene (see

Experimental procedures). Transgenic Arabidopsis con-

taining this ApL3::P450 construct survived the presence of

the R7402 proherbicide only under non-inducing sugar

conditions (Figure 2d). Screening conditions were opti-

mized for line A3P2, which contains a single site insertion

of the transgene on the long arm of chromosome 5

(approximately 5 cM from marker MBK5). M2 seeds from

an EMS mutagenized population (see Experimental pro-

cedures) were plated on media containing 100 mM sucrose

and 1 ng ml±1 R7402 proherbicide. Seedlings that showed

greening of the cotyledons after 7±10 days were trans-

ferred to plates with normal MS medium and allowed to

recover. The progeny of these putative mutants were re-

tested on the herbicide medium. From an initial screen of

40 000 M2 seeds, representing about 2000 M1 lines, we

isolated eight mutants that survived the screening condi-

tions. To select against mutants unrelated to the activity of

the ApL3 promoter, such as those defective in chloroplast

import or resistant to the herbicide, we tested the sugar

inducibility of the endogenous ApL3 gene. Sucrose induc-

tion of the endogenous ApL3 gene was reduced in four of

the mutants (Figure 1b), which we have named impaired

sucrose induction (isi) mutants.

The reduced sucrose induction of ApL3 expression was

maintained in twice back-crossed and re-selected lines.

The appearance of the four isi mutants did not change

Figure 1. Sucrose induction of ApL3 gene expression in wild-type and isimutants.(a) Sucrose induction of ApL3 expression in Arabidopsis wild-typeseedlings grown for 7 days in continuous light conditions, on MSmedium containing the indicated concentrations of sucrose (mM). Eachlane contains 10 mg of total RNA; the blot was re-probed for 18S rRNA.(b) Induction of ApL3 expression in the parental transgenic line A3P2 andisi mutants. Seedlings were grown for 7 days in continuous light on MSmedium containing 150 mM sucrose. Each lane contains 10 mg of totalRNA; the blot was re-probed for 18S rRNA.

Impaired sucrose-induction mutants 423


signi®cantly on back-crossing, and is shown in Figure 2(e).

Subtle leaf phenotypes that were genetically linked with

the isi phenotype were observed for isi1 and isi4 (data not

shown). The mature leaves of isi1 appeared more robust

and signi®cantly darker green in colour. Chlorophyll levels

were found to be elevated: typical values for greenhouse-

grown plants were 1.27 mg g±1 FW (SD 6 0.02) for the

A3P2 parental line, and 1.52 mg g±1 FW (SD 6 0.03) for the

isi1 mutant. The mature leaves of isi4 were slightly

greener, but smaller and narrower in shape, while the

overall stature of isi4 was also reduced. The isi2 and isi3

mutants were indistinguishable from the parental trans-

genic line.

Isi3 and isi4 are allelic to known sugar-response mutants

As observed for most other sugar-sensing screens, the

penetrance of the isi phenotype was not complete.

Greening of seedlings on the R7402 medium varied

between 5 and 40% for the different mutants, but was

clearly distinct from the parental A3P2 line (below 0.1%

greening; Figure 3). The isi mutants were further char-

acterized by studying their behaviour on sugar-sensing

screens that use germination and seedling establishment

(`greening') on high-sugar media. These studies, and their

chromosomal position in the Arabidopsis genome, were

used to determine possible allelism to known sugar-

response mutants.

On media containing 6% glucose, seedling establish-

ment is normally prevented. Mutants that green on 6%

glucose media are said to be glucose insensitive (gin; Zhou

et al., 1998). Plating the isi mutants on media with 6%

glucose showed that both isi3 and isi4 had a gin

phenotype. No such phenotype was observed for isi1

and isi2 (Figure 3). Other screens tested were mannose-

insensitive germination (mig, Pego et al., 2000), and seed-

Figure 2. GUS histochemistry of ApL3expression, sucrose-dependent survival ofline A3P2 on R7402 medium, andappearance of isi mutants.(a) Detached leaves of tissue culture-grownplants of line A3G5 were ¯oated on water(left) or 200 mM sucrose (right) for 24 h inthe dark. GUS staining was for 6 h, andtissues were cleared in 70% ethanol.(b) Thin section showing localization ofGUS staining to the starch sheath cell layerbelow the vascular bundle.(c) GUS staining of a developing seed: theembryo is at heart stage. Tissue was clearedin a chloralhydrate solution.(d) Survival of the transgenic line A3P2containing the ApL3::P450 constructdepends on sucrose concentration.Seedlings of line A3P2 were grown for7 days in continuous light on mediacontaining 1 ng ml±1 R7402 proherbicideand either 10 mM or 100 mM sucrose.(e) Appearance of the parental transgenicline A3P2 (middle), isi1 (top left), isi2 (topright), isi3 (bottom left) and isi4 (bottomright).



ling development on high-sucrose medium (Laby et al.,

2000; Pego et al., 2000). Of the isi mutants, only isi3 was

able to germinate on 7.5 mM mannose (data not shown).

Slightly different sucrose concentrations were used to

select mutants able to develop on high-sucrose medium

(Laby et al., 2000; Pego et al., 2000). On medium containing

350 mM sucrose, both isi3 and isi4 showed seedling

development (data not shown).

The isi mutants (in the Columbia ecotype) were mapped

by crossing to the Landsberg erecta ecotype (Ler). Mutants

were reselected in the F2 by plating on R7402 medium that

included kanamycin to select for the ApL3::P450 construct.

Mapping was with the use of both SSLP (Bell and Ecker,

1994) and CAPS (Konieczny and Ausubel, 1993) markers.

The behaviour of the mutants during mapping established

that all four mutants are recessive. isi1 was positioned on

the long arm of chromosome IV (between markers RPS2

and g8300), and isi2 on the top arm of chromosome I

(between nga63 and g2395). No known sugar-response

mutants map to these two positions; this, along with the

absence of additional sugar-sensing phenotypes, suggests

that these two mutants are speci®c to this particular

screen.

During mapping of isi3, close linkage was observed with

marker nga168 on chromosome II. This is similar to the

position described for the sucrose-uncoupled6 (sun6,

Dijkwel et al., 1997) mutant that was recently reported to

encode ABI4 (Huijser et al., 2000), an Apetala2-type tran-

scription factor originally isolated as the abscisic acid-

insensitive mutant abi4 (Finkelstein et al., 1998). Alleles of

abi4 have also been identi®ed in sugar-sensing screens

selecting for vegetative development on high-sugar media

(Arenas-Huertero et al., 2000; Laby et al., 2000). As shown

in Figure 3, isi3 also shows abscisic acid-insensitive

germination. Testing the F1 of crosses between isi3 and

abi4 on both 3 mM ABA and 6% glucose media showed that

these mutants belong to the same complementation group

(data not shown). Sequencing the abi4 gene in isi3

revealed the introduction of a stop codon at position 188

of the amino-acid sequence (a CAA to TAA conversion). In

an additional screen for isi mutants from a further 4000 M1

lines, a second isi3/abi4 allele was isolated. In this second

allele an amino-acid substitution of a highly conserved

residue in the Apetala2 domain was observed. The nega-

tively charged glutamic acid at position 69 was replaced by

a positively charged lysine (GAG to AAG). Laby et al. (2000)

reported the very same abi4 mutation in their sis5-4/abi4-

104 allele.

isi4 was mapped using both its survival on the R7402

proherbicide and its gin phenotype. A position on chromo-

some I was found 5 cM above the marker nga128. This

position is close, but not identical, to the position reported

for gin1 (0.5 cM from nga128; Zhou et al., 1998). However,

testing the F1 of crosses between isi4 and gin1-1 on media

containing 6% glucose showed that both mutations belong

to the same complementation group (Table 1). The use of

SSLP markers polymorphic for the Col (isi4) and WS (gin1-

1) ecotypes con®rmed the hybrid genotype of the F1 plants

(data not shown). Additional mapping of both isi4 and gin1

effectively localized both mutants to the BAC clone F19K6.

Recombinants closely ¯anking this BAC clone were

Figure 3. Seedling development of the isi mutants and control lines onmedia containing the R7402 proherbicide, 6% glucose or 3 mM ABA.Survival of isi mutants on media containing 100 mM sucrose and1 ng ml±1 R7402 was scored as the percentage of seedlings showinggreening after 10 days. This screen does not apply to the abi4 and gin1mutants as they do not contain the ApL3::P450 construct. The glucoseinsensitive phenotype was scored as the percentage of seedlingsgreening after 9 days in continuous light on media containing 6%glucose. Abscisic acid insensitive phenotypes were scored as thepercentage of greening seedlings after 5 days in continuous light onmedia containing 3 mM ABA. Values are the mean of three samplescontaining at least 100 seeds each (500 for R7402 scoring). Error barsrepresent standard deviations.

Table 1. Complementation tests between isi4, gin1-1 and aba2-1

Mutant line orF1/F2 seedsfrom crosses

Number ofseedlingsshowinggreening

Total numberof seedlings

Percentage ofseedlingsshowinggreening

A3P2 (Col-0) 0 153 0isi4 98 98 100gin1 83 85 98aba2 73 75 97isi4 3 gin1 F1 46 48 96isi4 3 aba2 F1 0 104 0isi4 3 aba2 F2 384 733 52aba2 3 gin1 F1 62 63 98

Wild-type, mutant, F1 and F2 seeds were grown on 6% glucosemedia for 7 days in continuous light; glucose insensitive pheno-types were scored as seedlings that showed greening.



obtained (data not shown), while no recombinants were

observed with marker F19K6.

Additional phenotypes characteristic for abscisic acid

de®ciency were observed in both isi4 and gin1-1, and an

interpretation of isi4/gin1 as a mutant with reduced ABA

levels is presented below. Several groups recently

reported that ABA-de®cient mutants show gin phenotypes

(Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby

et al., 2000). The ABA biosynthesis mutant aba2 has also

been mapped to the long arm of chromosome I using

classical markers (LeÂon-Kloosterziel et al., 1996).

Complementation testing of the isi4 and gin1-1 mutants

with aba2-1 resulted in apparently con¯icting results. The

F1 of crosses between aba2-1 and gin1-1 suggested that

they were allelic, and PCR con®rmed the hybrid genotype.

However, the F1 of crosses between isi4 and aba2-1

showed that these mutants were able to complement

each other (Table 1). Testing the F2 of the isi4 3 aba2-1

crosses showed a 1 : 1 segregation for the gin phenotype,

consistent with two closely linked independent muta-

tions. The possibility of inter-allelic complementation is

discussed below.

Sugar induction of starch biosynthetic genes is enhanced

by ABA

We further characterized the isi mutants by analysing the

sugar induction of starch biosynthetic genes in mature

leaves. Different methods were used to study sugar

regulation of gene expression in leaves. Sugars were fed

either via the petiole using the transpiration stream, or by

¯oating or submersing leaves or leaf discs in a sugar

solution (Krapp et al., 1991; MuÈ ller-RoÈ ber et al., 1990;

Takeda et al., 1994). We observed that the method of

sugar feeding resulted in different patterns of ApL3 gene

expression in the isi mutants (Figure 4). Petiole feeding, in

which leaves were detached by cutting the petioles under

water and placing them in a solution of 100 mM sucrose

for 24 h in the dark, resulted in a uniformly high induction

of the ApL3 gene. No differences were observed between

the parental transgenic control line and the isi mutants.

However, when similar detached leaves were submerged

in a 100 mM sucrose solution for 24 h in the dark, ApL3

expression was reduced in isi1 and isi2, was similar to the

control line in isi3, and was almost undetectable in isi4

(visible only after prolonged exposure). This behaviour

was not restricted to the ApL3 gene, but was also observed

for another sucrose-inducible starch biosynthetic gene,

Sbe2.2, encoding a starch-branching enzyme (Khoshnoodi

et al., 1998). A possible explanation for these results is that

the petiole-fed leaves have high water evaporation rates

and could be subject to dehydration stress (although they

were not severely wilted), which would lead to higher ABA

levels. This suggestion was supported by the fact that

RD22, a gene responsive to ABA and dehydration stress

(Yamaguchi-Shinozaki and Shinozaki, 1993), was highly

expressed in the petiole-fed leaves, while much lower

RD22 transcript levels were detected in submerged leaves

(Figure 4). Differences in RD22 expression were observed

between the different isi mutants. isi4, in particular,

showed a lower induction. These results, and the identi-

®cation of isi3 as an allele of the abscisic acid-insensitive

mutant abi4, led us to investigate the role of ABA in sugar

induction of starch biosynthetic genes.

Detached leaves of wild-type Arabidopsis plants were

placed in water or 100 mM sugar solution for 24 h in the

dark, in the absence or presence of 10 mM ABA (Figure 5a).

No induction of ApL3 expression was observed in water,

and ABA alone was unable to induce ApL3 expression. The

use of 100 mM mannitol as an osmotic control, either with

or without ABA, was also not effective. ApL3 expression

could be induced only in the presence of 100 mM sucrose.

This sucrose-induced ApL3 expression was greatly

enhanced in the presence of ABA. Although a basal level

of Sbe2.2 expression was present under all conditions

tested, Sbe2.2 transcript levels were increased in the

presence of sucrose. ABA was also able to further increase

sucrose-mediated increases in Sbe2.2 transcript levels.

The expression of the drought- and ABA-inducible RD22

gene showed the effectiveness of the ABA treatment.

As the 100 mM sucrose treatment was able to induce

RD22 expression slightly (Figure 5a), it was possible that a

more ef®cient uptake of sucrose resulted in ApL3 expres-

Figure 4. Sucrose induction of mature leaves of wild-type and isimutants.Leaves were detached from plants grown for 24 days in continuous light,and either petiole-fed or submersed with a 100 mM sucrose solution for24 h in the dark. The parental A3P2 transgenic line was used as control.Transcript levels were determined for the ApL3 gene, the starch-branching enzyme Sbe2.2, and the drought- and ABA-inducible RD22gene. Probing with the 18S rRNA probe served as a loading control. Eachlane contained 10 mg total RNA.



sion in direct response to osmotic signals. However, high

mannitol concentrations (300 mM), which were able to

induce osmotic responses such as RD22 expression, did

not induce the ApL3 gene (Figure 5b), while 50 mM

sucrose effectively induced ApL3 but not the RD22 gene.

The presence of 250 mM mannitol enhanced the sucrose-

mediated ApL3 induction. In conclusion, these results

suggest that sugar induces ApL3 expression and that ABA,

although ineffective by itself, is able signi®cantly to

enhance the response to sugar.

Isi4/gin1 shows characteristics of a mutant with reduced

ABA levels

The identi®cation of isi3 as an allele of abi4, and the results

of the sugar/ABA induction experiment (Figure 5a),

prompted further investigation of the isi mutants for

ABA-related phenotypes. Characteristics of ABA de®ciency

include excessive water loss due to impaired stomatal

functioning, and reduced seed dormancy. Measuring the

decrease over time in fresh weight of mature leaves after

detachment showed signi®cant water loss for isi4, and

severe water loss in the case of gin1-1 (Figure 6a,b). Water

loss in isi1 was slightly higher compared to the parental

control line, and was consistently observed in four inde-

pendent experiments. The rate of water loss observed for

isi2 and isi3 was similar to the A3P2 parental line (Figure

6a). Measuring seed dormancy further con®rmed an ABA-

de®ciency phenotype for isi4/gin1. Plating fresh seeds

directly on MS medium showed high germination rates for

both isi4 and gin1-1 (Figure 6c). In contrast to the water-

loss experiment, seed dormancy was normal for isi1 but

seemed reduced for isi2. The aba2-1 mutant was used as

control for ABA de®ciency in both types of experiment.

Further evidence for the interpretation of isi4/gin1 as an

ABA-de®cient mutant was obtained by measuring internal

ABA levels using an immuno-assay. Mature leaves from

plants grown under similar conditions to those used for

the water-loss experiment were harvested and extracted in

a 90% methanol solution (see Experimental procedures).

ABA levels were reduced in both isi4 and gin1-1, consistent

with the severity of their water-loss phenotype (Figure 6d).

Discussion

We used a genetic approach to study the factors involved

in sugar-induced gene expression of starch biosynthetic

genes in Arabidopsis. AGPase activity is a major control

point in starch biosynthesis, and regulation of enzyme

levels by gene expression also contributes to starch

accumulation (Sweetlove et al., 1999). The enzyme con-

sists of two small and two large subunits, and differential

expression of subunits is thought to generate isoenzymes

with distinct characteristics, such as differences in allos-

teric regulation, and with different kinetic properties (Doan

et al., 1999). The highly sugar-inducible third large subunit

(ApL3) of Arabidopsis was used to isolate mutants affect-

ing its transcriptional regulation. Its sequence homology

and tissue-speci®c expression in the starch sheath and

during seed development are consistent with its classi®-

cation as an Arabidopsis sink tissue-speci®c large subunit

Figure 5. ABA enhances sugar induction of ApL3 expression.(a) Detached leaves of wild-type Arabidopsis plants grown for 24 days incontinuous light were submersed in either water (H2O), 100 mM mannitol(Man) or 100 mM sucrose (Suc) in the absence or presence of 10 mM ABA(+ABA) for 24 h in the dark. Transcript levels were determined for theApL3 gene, the starch-branching enzyme Sbe2.2, and the drought- andABA-inducible RD22 gene. Probing with the 18S rRNA probe served as aloading control. Each lane contained 10 mg total RNA.(b) Leaf strips of wild-type Arabidopsis plants grown for 24 days incontinuous light were ¯oated on either water (H2O), 50 mM sucrose(Suc), 300 mM mannitol (Man), or a combination of 50 mM sucrose and250 mM mannitol (Suc + Man) for 24 h in the dark. Gene expression wasdetermined as described for (a).



(du Jardin et al., 1997; Park and Chung, 1998; Smith-White

and Preiss, 1992).

To isolate mutants with reduced sugar induction of the

ApL3 promoter, we used a bacterial cytochrome P450 gene

as a negative selection marker (O'Keefe et al., 1994). The

requirement for chloroplast targeting of the cytochrome

P450 (to obtain electrons from ferredoxin) results in the

additional isolation of putative chloroplast import

mutants. These and other by-products, such as resistance

to the herbicide, were easily distinguished from mutants

affecting ApL3 expression in trans by determining the

expression of the endogenous ApL3 gene. The P450-R7402

system has previously been used in Arabidopsis as a

counter-selection marker during the generation of a dSpm

transposon-insertion collection (Tissier et al., 1999), but to

our knowledge this is its ®rst use in a genetic screen.

Two of the isi mutants isolated (isi3 and isi4) were found

to be allelic to mutants identi®ed in sugar-sensing screens

based on seedling development (Arenas-Huertero et al.,

2000; Huijser et al., 2000; Laby et al., 2000). These groups

reported that the sun6, sis5 and gin6 mutants were alleles

of abscisic acid insensitive4, and that known aba and abi

mutants exhibited glucose insensitive phenotypes. Here

we present evidence that gin1 itself is ABA de®cient,

showing characteristics such as defective stomatal closure

Figure 6. Stomatal closure, seed dormancy and ABA concentrations inthe isi mutants, gin1-1 and aba2-1.(a) Stomatal closure of the isi mutants was determined by the reductionin fresh weight (% FW loss) of detached leaves over time (min). Valuesare mean of three samples containing four mature leaves each. Errorbars represent standard deviations. Plants were grown in soil undergreenhouse conditions for 4 weeks and placed under a translucent cover24 h prior to leaf detachment. Line A3P2 is the parental transgenic line.(b) Loss of stomatal closure in gin1-1 (in Ws ecotype). isi4 and its parentline A3P2 are given for comparison. Ws represents the Wassilewskijagenetic background of gin1-1; aba2 is in the Columbia ecotype.Conditions as described for (a).(c) Seed dormancy in the isi mutants, gin1-1 and aba2. Freshly harvestedseeds (approximately 20 days after anthesis) were sterilized and directlyplated on MS media. Seed germination was scored as the percentage ofseeds showing radicle emergence after 4 days. Values are the mean ofthree samples containing at least 100 seeds each. Error bars representstandard deviations.(d) Concentration of free ABA in the isi mutants, gin1-1 and aba2 andtheir control lines. Mature leaves were harvested as described for (a) andwere extracted in 90% methanol. ABA levels were determined using acompetitive ELISA. Values are mean of two separate extractions, errorbars represent standard deviations.



and reduced seed dormancy. Our complementation data

(Table 1) show the apparent contradiction that while gin1-

1 is allelic to both isi4 and aba2-1, the latter two mutants

complement each other. Preliminary results suggest that

an explanation can be found in the identity of the encoded

gene and the nature of the mutations in the different

mutants. Our mapping of isi4 effectively localizes it to the

BAC clone F19K6. In both isi4 (A236 to V236) and aba2-1

(S264 to N264), single amino-acid changes were found in the

coding region of a member of the short-chain dehydro-

genase/reductase gene family (gene At1g52340) located on

this BAC. PCR suggested that in gin1-1 a substantial part of

this gene is deleted. The multimeric nature of this class of

enzymes allows for the possibility of interallelic comple-

mentation. A combination of the full-size polypeptides

encoded in aba2-1 and isi4 could constitute a biologically

functional enzyme. Although this interallelic complemen-

tation hypothesis requires further testing, the alternative

hypothesis ± that the possible chromosomal defect in gin1-

1 affects two closely linked ABA biosynthetic genes ± is

less likely. The latter possibility would not explain the fact

that changes in the gene sequence of At1g52340 are

observed in all three mutants. Biochemical data are

required to con®rm the proposed role of this short-chain

dehydrogenase/reductase in ABA biosynthesis as the

xanthoxin oxidase suggested by Schwartz et al. (1997).

The gin1 mutant was originally interpreted as revealing

cross-talk with the ethylene signalling pathway because it

could be phenocopied by ethylene precursor treatment of

wild-type plants, or by constitutive ethylene biosynthesis

and constitutive ethylene-signalling mutants (Zhou et al.,

1998). A plausible explanation for these ethylene effects

and our ABA de®ciency data can be found in recent reports

that ethylene appears to be a negative regulator of ABA

action during germination (Beaudoin et al., 2000;

Ghassemian et al., 2000).

In addition to the abi4 (isi3) and gin1/aba2 (isi4) alleles,

two new sugar-response mutants, isi1 and isi2, were

isolated, which are involved in the co-ordinated sugar

induction of two starch biosynthetic genes.

Characterization of these two mutants showed that they

are speci®c to our screen, as they do not show additional

phenotypes on any of the germination screens using high-

sugar media. They could be part of a sugar signal-speci®c

pathway, and the observed increase in chlorophyll levels

in isi1 suggests that not only the expression of starch

biosynthetic genes is affected. Preliminary analysis of an

isi1/isi4 double mutant shows a highly additive phenotype

with almost 100% survival on the herbicide medium

(unpublished data). But as the stomatal closure phenotype

of isi4 is less severe than either gin1-1 or aba2-1, it

probably has some residual function. It is therefore not

possible to determine if isi1 and isi4 belong to the same or

to interacting genetic pathways. The model in Figure 7

places isi1 and isi2 in a pathway separate from ABA

signalling, but molecular analysis of the genes responsible

is required to con®rm this.

Our data provide genetic evidence for the involvement

of ABA signalling in the co-ordinated sugar induction of

two starch-biosynthetic genes. RNA gel-blot analysis

showed that ABA by itself was unable to induce the two

starch-biosynthetic genes, but greatly enhanced their

induction by sugar. Based on these observations, we

propose a model in which ABA signalling modulates

responses to a separate sugar signal (Figure 7). The

differences in ApL3 induction when comparing petiole

feeding with leaf submersion showed that ABA may be of

more importance than the actual sugar concentration in

determining the level of response. In the absence of ABA

signalling (e.g. ApL3 expression in isi4 when submersed in

100 mM sucrose; Figure 4), sugar induction of ApL3

expression is severely reduced. The importance of ABA

and ABI4 for sugar responses has also been observed by

groups studying sugar repression of seedling establish-

ment and developmentally induced photosynthetic gene

expression (Arenas-Huertero et al., 2000; Huijser et al.,

2000; Laby et al., 2000).

Figure 7. Model in which ABA and other plant hormones modulateresponses to sugar signals.ABA signalling induces a metabolic state (storage mode) in whichstorage-related processes are more sensitive to a separate sugar signal.ABI4 (isi3) is part of this ABA-signalling pathway, and in its absence amobilization mode becomes dominant. Gin1/isi4/aba2 is involved in ABAbiosynthesis. ISI1 and ISI2 may be part of a speci®c sugar-signallingpathway. High osmotic conditions, including high sugar, result in ABAproduction and higher sensitivity to the sugar signal. Other hormonessuch as gibberellins may promote the mobilization mode. See text fordetailed description of the model.



SoÈ derman et al. (2000) recently reported that ABI4 is a

transcriptional activator, expressed most strongly in

seeds, but also at low levels in vegetative tissues. Over-

expression of ABI4 resulted in the ABA-dependent expres-

sion of late embryogenesis-related genes in vegetative

tissues (SoÈ derman et al., 2000). These expression data are

consistent with our ®nding that the effect of our isi3 allele

of abi4 was most pronounced at the seedling stage.

Among the isi mutants tested, the isi3 mutant showed

the highest survival on the R7402 selection medium.

Although ApL3 expression was similar to wild type in

sugar-fed mature isi3 leaves, Sbe2.2 expression was

reduced, suggesting some role for ABI4 in mature plants.

Previous physiological experiments on feedback inhibition

of photosynthesis in the sun6 allele of abi4 also suggested

a role for ABI4 in mature plants (van Oosten et al., 1997).

Mutants in abi4 have now been isolated in a number of

genetic screens, all involving selection at the seedling

stage. Originally isolated as a mutant germinating in the

presence of ABA (Finkelstein et al., 1998), it also shows

germination and seedling establishment on high glucose,

sucrose and mannose (Arenas-Huertero et al., 2000;

Huijser et al., 2000; Laby et al., 2000; this study), and high

salinity and osmolarity (Quesada et al., 2000). This sug-

gests that in abi4 mutants, a variety of external stimuli no

longer inhibit a program of seedling establishment. ABI4

could be part of an ABA-signalling pathway that confers a

particular metabolic state on a tissue (Figure 7). In this

`storage mode', photosynthetic genes may be more sen-

sitive to sugar repression, while genes related to storage

functions, such as AGPase, are more easily induced by

high sugar. In the absence of a functional abi4 gene, a

`mobilization mode' might dominate, resulting in the

preferential activation of growth- and development-related

programs such as seedling establishment, even when

grown on high-sugar media (Figure 7).

Arenas-Huertero et al. (2000) recently proposed a model

in which ABA and ABI4 acted downstream of a hexokinase-

mediated sugar signal. Plants overexpressing the

Arabidopsis hexokinase gene AtHXK1 resulting in glucose

hypersensitivity, and containing the gin5 mutation confer-

ring ABA de®ciency, still displayed a glucose-insensitive

phenotype. Zhou et al. (1998) reported similar results for

plants containing the gin1 mutation and the 35S±AtHXK1

construct. These results were interpreted as ABA being

epistatic to the sugar signal in the same pathway, but in

our view are also consistent with a model in which the

response to the sugar signal largely depends on the

presence of a separate ABA signal (Figure 7). Moreover,

these previous interpretations presuppose that the sugar

signal is not saturated on the high-glucose media used

(330 mM), and can be enhanced by hexokinase over-

expression. Their observation that the application of ABA

to wild-type plants made them hypersensitive to glucose,

resulting in arrest of seedling development at lower

glucose concentrations (Arenas-Huertero et al., 2000), is

also consistent with our model. The same authors

observed an increase in ABA levels on high-sugar media

(7% glucose), and noted that this could also be attributed

to osmotic stress. They hypothesized that as osmotic

stress alone did not lead to seedling developmental arrest,

at least one other independent component is required in

addition to ABA to promote glucose-dependent develop-

mental arrest (Arenas-Huertero et al., 2000). Our model

does not require an additional unknown component. We

propose that the effect of high-sugar media may be

twofold, with increased ABA levels as a result of osmotic

effects making the seedlings more sensitive to the same

sugar functioning as a signal (Figure 7). In this view,

seedling developmental arrest on high-sugar media is the

consequence of an osmotically induced `storage mode'

preventing mobilization and growth. Indeed, Laby et al.

(2000) observed arrest of vegetative development when

400 mM sorbitol was combined with only 28 mM glucose.

Their alleles of aba2 (sis4) and abi4 (sis5) were insensitive

to this combination of high osmolarity and low sugar. This

observation provides an explanation for the isolation of

abi4 alleles in screens based on high salinity and

osmolarity (Quesada et al., 2000). Osmotic effects also

appeared to enhance the sugar induction of ApL3 expres-

sion (Figure 5b).

Our model also proposes a mobilization mode which

could be promoted by other hormone-signalling pathways

such as gibberellins (Figure 7). Induction of a-amylase

expression by gibberellins (GA), and repression by sugar

signalling and abscisic acid, have been well documented

during seed germination in cereals (Jacobsen and Beach,

1985; Perata et al., 1997; Smeekens, 2000). The effects of

GA and ABA may be explained by opposing effects on

sensitivity to a putative sugar signal. It is noteworthy that

promoter analysis of sugar-responsive a-amylases has

indicated that GA, ABA and sugar-responsive elements

appear to overlap, suggesting that the signal-transduction

pathways communicate at a point upstream of the pro-

moter elements (reviewed by Smeekens, 2000). Possible

mechanisms for ABA modulation of sugar signalling are

suggested by the observation that the SNF1-related kinase

PKABA1 is induced in barley aleurone layers by ABA, and

that its constitutive expression drastically suppressed a-

amylase expression (GoÂ mez-Cadenas et al., 1999). In

Arabidopsis, the requirement for GA during seed germin-

ation has been used to isolate ABA-de®cient mutants

(LeÂon-Kloosterziel et al., 1996) and, consistent with the

predictions of our model, abi4 mutants are able to

germinate in the presence of the gibberellin biosynthesis

inhibitor paclobutrazol (Laby et al., 2000). In the absence of

ABA signalling promoting a storage mode, less activation

of a mobilization mode by gibberellins may be required.



Hormonal control of sugar signalling may be a mechan-

ism to regulate sugar-responsive gene expression in a way

that is speci®c for a certain tissue or developmental stage,

or in response to a particular environmental stimulus.

Sugar concentrations vary greatly in plants, both between

different tissues and during the diurnal cycle, as a result of

photosynthesis and carbohydrate partitioning. Hormone

balances could determine the way tissues respond to these

changing sugar concentrations, for example by partitioning

carbohydrate into growth and metabolism, or into storage.

Dissecting and separating the complex interactions

between hormone and sugar signals will further our

understanding of how plants partition their carbohydrates.

Experimental procedures

Plant material and growth conditions

Wild-type and transgenic Arabidopsis thaliana plants were in theColumbia ecotype, the aba2-1 and abi4 mutants were also in theColumbia background and were obtained from the NottinghamArabidopsis Stock Centre (NASC, Nottingham, UK). The gin1-1mutant was kindly provided by Dr Li Zhou (MassachusettsGeneral Hospital, Boston, MA, USA) and in the Wassilewskija(Ws) ecotype. The wild-type Ws ecotype line was derived from theFeldmann T-DNA collection, also obtained from the NASC.

For in vitro-grown plants, seeds were surface-sterilized, im-bibed at 4°C for at least 3 days, and plated on MS medium(Duchefa Biochemie BV, Haarlem, The Netherlands) containing1% glucose. To determine ABA-insensitive germination, seedswere plated on sugar-free medium containing 3 mM ABA (mixedisomers; Sigma, Poole, Dorset, UK) under continuous light condi-tions. Plants for stomatal closure experiments and ABA measure-ment were grown in the greenhouse for about 4 weeks. Plantswere well watered, placed under a transparent cover, and trans-ferred to controlled growth conditions (22°C, 16 h light) 24 hbefore the start of the experiments. For determination of seeddormancy, ¯owers were selected at anthesis and the adjacentdeveloping siliques and ¯ower buds were removed. Seeds wereharvested after 20 days, surface sterilized and plated immediately.

Isolation of ApL3 genomic and cDNA sequences

To isolate a genomic clone of the ApL3 gene, high-density colony®lters of the TAMU and IGF BAC libraries (Bent et al., 1998) werescreened using an ApL3 cDNA fragment (Villand et al., 1993) asprobe. A 6 kb EcoRI fragment from BAC TAMU22F8 containingthe ApL3 gene was subcloned into pBluescript KS (Stratagene,La Jolla, CA, USA) and sequenced using BigDyeTerminator RRMix (PE Applied Biosystems, Warrington, UK). The predicted exonstructure of the ApL3 gene was con®rmed by RT±PCR isolation ofa near full-length ApL3 cDNA fragment. RT±PCR was on total RNAfrom sucrose-induced leaves, using the gene-speci®c primersAPL3AS: 5¢-AAGATGGATTCTTGTTGCAACTTTAGCTTGG-3¢ andAPL3AP: 5¢-GTGGTTGTCGAAAAGGCCACCATTAAAGACG-3¢. TheApL3 cDNA fragment was cloned in the pGEM-T vector (Promega,Madison, WI, USA), and sequenced.

Transgenic lines and GUS histochemistry

The GUS reporter gene was from plasmid pRAJ275 (Jeffersonet al., 1987) and contained an NcoI site on the ATG start codon.

The cytochrome P450 negative selection marker was subcloned asa PstI fragment from plasmid pSSU-SU121 (O'Keefe et al., 1994)into pBluescript KS. The linker oligo 5¢-CTAGACCATGGCTTC-CTCTGTGATTTCCAGTGCA-3¢ restored the sequence of the petu-nia RBCS transit peptide while introducing an NcoI site on theATG start codon. The ApL3 reporter gene constructs were madeusing a PCR-based cloning method. Using primer APL3Nco: 5¢-GAATCCATGGTTTTTTAGCTGGAATGAGACAAG-3¢, a 244 bp PstI/NcoI 5¢ fragment was generated with the NcoI site on the ApL3start codon. Using primer APL3Eco: 5¢-CGGAATTCACAAAACGA-TCTCAAGACCGC-3¢, a 149 bp EcoRI/HindIII 3¢ fragment wasgenerated, introducing an EcoRI site after the ApL3 stop codon.The sequence of both PCR-generated fragments was con®rmed.The PCR fragments were ligated on either side of the reportergenes. Additional 5¢ and 3¢ sequences were added to the reportergene constructs, resulting in 4.5 kb of the 5¢ region and 1.5 kb ofthe 3¢ region ¯anking the reporter genes. The ApL3::GUS andApL3::P450 constructs were cloned in the BamHI and KpnI sites ofthe plant-transformation vector pGreen0029 (Hellens et al., 2000).Arabidopsis transformation was by vacuum in®ltration usingAgrobacterium strain GV3101 (pMP90). Transformants wereselected on kanamycin (50 mg ml±1)-containing medium. Linessegregating for single insertion sites were selected. GUShistochemistry and microscopy were performed as describedpreviously (Rook et al., 1998).

Isolation and genetic mapping of isi mutants

From the ApL3::P450 transformants, line A3P2 was selected formutagenesis and seeds were bulked up. Approximately 20 000seeds were treated with a 0.5% (40 mM) solution of ethylmethanesulfonate (EMS; Sigma) for 6 h. Mutagenized M1 seedswere sown in pools on soil and harvested. M2 seeds were surface-sterilized and selected on MS medium containing 100 mM

sucrose and 1 ng ml±1 of the R7402 proherbicide (2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl) aminocarbonyl]-1,2-benzoisothiazole-7-sulfonamide-1,1-dioxide; provided byDuPont, Wilmington, DE, USA). Seedlings were grown for 7±10 days in continuous light, and greening seedlings were trans-ferred to proherbicide-free medium to recover. After 3±4 weeksthe plants were transferred to soil and the progeny collected forre-testing on the R7402 selection medium. A total of 150 000 M2

seeds, representing about 6000 M1 lines, were screened. Aftercon®rmation of herbicide survival, the sugar induction of theendogenous ApL3 gene was determined by RNA gel-blot analy-sis.

The isi mutants were mapped using SSLP (Bell and Ecker, 1994)and CAPS (Konieczny and Ausubel, 1993) markers polymorphicbetween the Col and Ler Arabidopsis ecotypes. At least 100chromosomes were tested for each mutant, with 800 chromo-somes for the ®ne mapping of isi4, and 200 for the mapping ofgin1, which was crossed to both Col and Ler. Primers for thederived CAPS marker F19K6 were F19K6F: 5¢-CAAAATTAAA-AGTATATGTAATTG TTGAG-3¢ and F19K6R: 5¢-AGAAAATGGGA-AAGACTTACCTCGA-3¢; digestion was with XhoI.

RNA gel-blot analysis

RNA isolation was by grinding tissues in liquid nitrogen andextraction using TRIZOL reagent (Life Technologies, Paisley, UK)according to the manufacturer's protocol. RNA was dissolved in0.1% SDS and quanti®ed spectrophotometrically. RNA gelelectrophoresis and blotting was as described (Rook et al.,



1998). Gene-speci®c probes were digoxigenin-labelled (DIG-dUTP) by PCR using PCR DIG Labelling Mix (Roche Diagnostics,Lewes, UK) and gene-speci®c primers. Hybridization was asdescribed (Rook et al., 1998); washes were with 0.2 3 SSC, 0.1%SDS at 65°C, twice for 15 min. Detection was with anti-dig-oxigenin-AP, Fab fragments and the chemiluminescent substrateCSPD, according to the manufacturer (Roche). The ApL3 cDNAfragment in pGEM-T was used as template to generate an ApL3gene-speci®c probe using primers APL3AP and APL3AS. Theprobe for the Sbe2.2 gene was isolated by RT±PCR using thegene-speci®c primers SBE2A 5¢-GTTCTCTTACTCCACGCTTCA-CTC-3¢ and SBE2B 5¢-GTTCTACACGGTGCATAGACCATG-3¢.Primers RD22A: 5¢-ATGGCGATTCGGCTTCCTCTGATC-3¢ andRD22B: 5¢-CTAGTAGCTGAACCACACAACATG-3¢ were used toPCR amplify the RD22 coding region from genomic DNA. The18S rRNA probe was labelled using the T7 Quick prime kit(Pharmacia, Uppsala, Sweden) and a32P-dCTP.

Abscisic acid and chlorophyll measurements

Plants used for determining ABA concentrations were grown andtreated as described for the stomatal closure experiments. Leaveswere harvested and immediately frozen in liquid N2.Approximately 30 mg of grounded tissue was extracted in 1 mlof a 90% methanol solution, containing 20 ml l±1 acetic acid and10 mg l±1 butylated hydroxytoluene (BHT, Sigma) for 24 h at 4°C.Free ABA concentrations were measured by competitive ELISAusing the Phytodetek ABA kit (Agdia Inc., Elkhart, IN, USA)according to the manufacturer's protocol. Mixed ABA isomers(Sigma) were used as standard.

Leaves for chlorophyll measurements were frozen in liquid N2.Approximately 10 mg of grounded tissue was extracted in 1 ml80% (v/v) acetone and quanti®ed according to Arnon (1949).

Acknowledgements

We thank Dr Leszek Kleczkowski for providing the ApL3 cDNAfragment, Dr Ian Bancroft for providing the high-density BAClibrary ®lters, Dr Li Zhou for the gift of the gin1-1 seeds, and theNottingham Arabidopsis Stock Centre for the abi4 and aba2-1seeds. We thank E.I. du Pont de Nemours and Company (DuPont)for making available to us the R7402-dependent, cytochrome P450

conditional lethal marker system. Dr Margarete Baier is acknow-ledged for useful comments on the manuscript. This work wassupported by the European Community (Grant No. BIO4-CT96-0311). F.R. received a Marie Curie Fellowship from the EuropeanCommunity (Grant No. BIO4-CT98-5098).

References

Arenas-Huertero, F., Arroyo, A., Zhou, L., Sheen, J. and LeoÂ n, P.(2000) Analysis of Arabidopsis glucose insensitive mutants,gin5 and gin6, reveals a central role of the plant hormone ABAin the regulation of plant vegetative development by sugar.Genes Dev. 14, 2085±2096.

Arnon, D.J. (1949) Copper enzymes in isolated chloroplasts. PlantPhysiol. 24, 1±15.

Bae, J.M. and Liu, J.R. (1997) Molecular cloning andcharacterisation of two novel isoforms of the small subunit ofADPglucose pyrophosphorylase from sweet potato. Mol. Gen.Genet. 254, 179±185.

Beaudoin, N., Serizet, C., Gosti, F. and Giraudat, J. (2000)

Interactions between abscisic acid and ethylene signalingcascades. Plant Cell, 12, 1103±1115.

Bell, C.J. and Ecker, J.R. (1994) Assignment of 30 microsatelliteloci to the linkage map of Arabidopsis. Genomics, 19, 137±144.

Bent, E., Johnson, S. and Bancroft, I. (1998) BAC representation oftwo low-copy regions of the genome of Arabidopsis thaliana.Plant J. 13, 849±855.

Bhalerao, R.P., Salchert, K., BakoÂ , L., OÈ kreÂ sz, L., Szabados, L.,Muranaka, T., Machida, Y., Schell, J. and Koncz, C. (1999)Regulatory interaction of PRL1 WD protein with ArabidopsisSNF1-like protein kinases. Proc. Natl Acad. Sci. USA, 96,5322±5327.

Dijkwel, P.P., Huijser, C., Weisbeek, P.J., Chua, N.-H. andSmeekens, S.C.M. (1997) Sucrose control of phytochrome Asignaling in Arabidopsis. Plant Cell, 9, 583±595.

Doan, D.N.P., Rudi, H. and Olsen, O.-A. (1999) The allostericallyunregulated isoform of ADP-glucose pyrophosphorylase frombarley endosperm is the most likely source of ADP-glucoseincorporated into endosperm starch. Plant Physiol. 121,965±975.

du Jardin, P., Harvengt, L., Kirsch, F., Le, V.-Q., Nguyen-Quoc, B.and Yelle, S. (1997) Sink-cell-speci®c activity of a potato ADP-glucose pyrophosphorylase B-subunit promoter in transgenicpotato and tomato plants. Planta, 203, 133±139.

Finkelstein, R.R., Wang, M.L., Lynch, T.J., Rao, S. and Goodman,H.M. (1998) The Arabidopsis abscisic acid response locus abi4encodes an APETALA2 domain protein. Plant Cell, 10,1043±1054.

Ghassemian, M., Nambara, E., Cutler, S., Kawaide, H., Kamiya, Y.and McCourt, P. (2000) Regulation of abscisic acid signaling bythe ethylene response pathway in Arabidopsis. Plant Cell, 12,1117±1126.

GoÂ mez-Cadenas, A., Verhey, S.D., Holappa, L.D., Shen, Q., Ho, T.-H.D. and Walker-Simmons, M.K. (1999) An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers. Proc.Natl Acad. Sci. USA, 96, 1767±1772.

Halford, N.G. and Hardie, D.G. (1998) SNF1-related proteinkinases: global regulators of carbon metabolism in plants?Plant Mol. Biol. 37, 735±748.

Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S. andMullineaux, P.M. (2000) pGreen: a versatile and ¯exible binaryTi vector for Agrobacterium-mediated plant transformation.Plant Mol. Biol. 42, 819±832.

Herbers, K. and Sonnewald, U. (1998) Molecular determinants ofsink strength. Curr. Opinion Plant Biol. 1, 207±216.

Huijser, C., Kortstee, A., Pego, J., Weisbeek, P., Wisman, E. andSmeekens, S. (2000) The Arabidopsis sucrose uncoupled-6gene is identical to abscisic acid insensitive-4: involvement ofabscisic acid in sugar responses. Plant J. 23, 577±586.

Jacobsen, J.V. and Beach, L.R. (1985) Control of transcription of a-amylase and rRNA genes in barley aleurone protoplasts bygibberellin and abscisic acid. Nature, 316, 275±277.

Jang, J.-C., LeoÂ n, P., Zhou, L. and Sheen, J. (1997) Hexokinase asa sugar sensor in higher plants. Plant Cell, 9, 5±19.

Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUSfusions: b-glucuronidase as a sensitive and versatile genefusion marker in higher plants. EMBO J. 6, 3901±3907.

Khoshnoodi, J., Larsson, C.-T., Larsson, H. and Rask, L. (1998)Differential accumulation of Arabidopsis thaliana Sbe2.1 andSbe2.2 transcripts in response to light. Plant Sci. 135, 183±193.

Koch, K.E. (1996) Carbohydrate-modulated gene expression inplants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 509±540.

Konieczny, A. and Ausubel, F.M. (1993) A procedure for mapping



Arabidopsis mutations using co-dominant ecotype-speci®cPCR-based markers. Plant J. 4, 403±410.

Koûmann, J., Visser, R.G.F., MuÈ ller-RoÈ ber, B., Willmitzer, L. andSonnewald, U. (1991) Cloning and expression analysis of apotato cDNA that encodes branching enzyme: evidence for co-expression of starch biosynthetic genes. Mol. Gen. Genet. 230,39±44.

Krapp, A., Quick, P. and Stitt, M. (1991) Ribulose-1,5-bisphosphate carboxylase-oxygenase, other Calvin-cycleenzymes, and chlorophyll decrease when glucose is suppliedto mature spinach leaves via the transpiration stream. Planta,186, 58±69.

Krapp, A., Hofmann, B., SchaÈ fer, C. and Stitt, M. (1993) Regulationof the expression of rbcS and other photosynthetic genes bycarbohydrates: a mechanism for the `sink regulation' ofphotosynthesis? Plant J. 3, 817±828.

Laby, R.J., Kincaid, M.S., Kim, D. and Gibson, S.I. (2000) TheArabidopsis sugar-insensitive mutants sis4 and sis5 aredefective in abscisic acid synthesis and response. Plant J. 23,587±596.

LeÂon-Kloosterziel, K.M., Alvarez Gil, M., Ruijs, G.J., Jacobsen,S.E., Olszewski, N.E., Schwartz, S.H., Zeevaart, J.A.D. andKoornneef, M. (1996) Isolation and characterization of abscisicacid-de®cient Arabidopsis mutants at two new loci. Plant J. 10,655±661.

Martin, T., Hellmann, H., Schmidt, R., Willmitzer, L. and Frommer,W.B. (1997) Identi®cation of mutants in metabolically regulatedgene expression. Plant J. 11, 53±62.

Mita, S., Hirano, H. and Nakamura, K. (1997a) Negative regulationin the expression of a sugar-inducible gene in Arabidopsisthaliana. A recessive mutation causing enhanced expression ofa gene for b-amylase. Plant Physiol. 114, 575±582.

Mita, S., Murano, N., Akaike, M. and Nakamura, K. (1997b)Mutants of Arabidopsis thaliana with pleiotropic effects on theexpression of the gene for b-amylase and on the accumulationof anthocyanin that are inducible by sugars. Plant J. 11,841±851.

Morell, M.K., Bloom, M., Knowles, V. and Preiss, J. (1987) Subunitstructure of spinach leaf ADP-glucose pyrophosphorylase.Plant Physiol. 85, 182±187.

MuÈ ller-RoÈ ber, B.T., Koûmann, J., Hannah, L.C., Willmitzer, L. andSonnewald, U. (1990) One of two different ADP-glucosepyrophosphorylase genes from potato responds strongly toelevated levels of sucrose. Mol. Gen. Genet. 224, 136±146.

MuÈ ller-RoÈ ber, B., La Cognata, U., Sonnewald, U. and Willmitzer,L. (1994) A truncated version of an ADP-glucosepyrophosphorylase promoter from potato speci®es guard cell-selective expression in transgenic plants. Plant Cell, 6, 601±612.

NeÂmeth, K., Salchert, K., Putnoky, P. et al. (1998) Pleiotropiccontrol of glucose and hormone responses by PRL1, a nuclearWD protein in Arabidopsis. Genes Dev. 12, 3059±3073.

O'Keefe, D.P., Tepperman, J.M., Dean, C., Leto, K.J., Erbes, D.L.and Odell, J.T. (1994) Plant expression of a bacterialcytochrome P450 that catalyzes activation of a sulfonylureapro-herbicide. Plant Physiol. 105, 473±482.

Park, S.-W. and Chung, W.-I. (1998) Molecular cloning and organ-speci®c expression of three isoforms of tomato ADP-glucosepyrophosphorylase gene. Gene, 206, 215±221.

Pego, J.V., Kortstee, A.J., Huijser, C. and Smeekens, S.C.M. (2000)Photosynthesis, sugars and the regulation of gene expression.J. Exp. Bot. 51, 407±416.

Perata, P., Matsukura, C., Vernieri, P. and Yamaguchi, J. (1997)Sugar repression of a gibberellin-dependent signaling pathwayin barley embryos. Plant Cell, 9, 2197±2208.

Purcell, P.C., Smith, A.M. and Halford, N.G. (1998) Antisenseexpression of a sucrose non-fermenting-1-related proteinkinase sequence in potato results in decreased expression ofsucrose synthase in tubers and loss of sucrose-inducibility ofsucrose synthase transcripts in leaves. Plant J. 14, 195±202.

Quesada, V., Ponce, M.R. and Micol, J.L. (2000) Genetic analysisof salt-tolerant mutants in Arabidopsis thaliana. Genetics, 154,421±436.

Rook, F., Gerrits, N., Kortstee, A., van Kampen, M., Borrias, M.,Weisbeek, P. and Smeekens, S. (1998) Sucrose-speci®csignalling represses translation of the Arabidopsis ATB2 bZIPtranscription factor gene. Plant J. 15, 253±263.

Schwartz, S.H., LeÂon-Kloosterziel, K.M., Koornneef, M. andZeevaart, J.A.D. (1997) Biochemical characterization of theaba2 and aba3 mutants in Arabidopsis thaliana. Plant Physiol.114, 161±166.

Smeekens, S. (2000) Sugar-induced signal transduction in plants.Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 49±81.

Smeekens, S. and Rook, F. (1997) Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiol. 115, 7±13.

Smith-White, B.J. and Preiss, J. (1992) Comparison of proteins ofADP-glucose pyrophosphorylase from diverse sources. J. Mol.Evol. 34, 449±464.

SoÈ derman, E.M., Brocard, I.M., Lynch, T.J. and Finkelstein, R.R.(2000) Regulation and function of the Arabidopsis ABA-insensitive4 gene in seed and abscisic acid responsesignaling networks. Plant Phys. 124, 1752±1765.

Sokolov, L.N., Dejardin, A. and Kleczkowski, L.A. (1998) Sugarsand light/dark exposure trigger differential regulation of ADP-glucose pyrophosphorylase genes in Arabidopsis thaliana.Biochem. J. 336, 681±687.

Stark, D.M., Timmerman, K.P., Barry, G.F., Preiss, J. and Kishore,G.M. (1992) Regulation of the amount of starch in plant tissuesby ADP glucose pyrophosphorylase. Science, 258, 287±292.

Sweetlove, L.J., MuÈ ller-RoÈ ber, B., Willmitzer, L. and Hill, S.A.(1999) The contribution of adenosine 5¢-diphosphoglucosepyrophosphorylase to the control of starch synthesis inpotato tubers. Planta, 209, 330±337.

Takeda, S., Mano, S., Ohto, M. and Nakamura, K. (1994) Inhibitorsof protein phosphatases 1 and 2A block the sugar-induciblegene expression in plants. Plant Physiol. 106, 567±574.

Tissier, A.F., Marillonnet, S., Klimyuk, V., Patel, K., Angel Torres,M., Murphy, G. and Jones, J.D.G. (1999) Multiple independentdefective Suppressor-mutator transposon insertions inArabidopsis: a tool for functional genomics. Plant Cell, 11,1841±1852.

van Oosten, J.J., Gerbaud, A., Huijser, C., Dijkwel, P.P., Chua, N.-H. and Smeekens, S.C.M. (1997) An Arabidopsis mutantshowing reduced feedback inhibition of photosynthesis. PlantJ. 12, 1011±1020.

Villand, P., Olsen, O.-A. and Kleczkowski, L.A. (1993) Molecularcharacterization of multiple cDNA clones for ADP-glucosepyrophosphorylase from Arabidopsis thaliana. Plant Mol. Biol.23, 1279±1284.

Yamaguchi-Shinozaki, K. and Shinozaki, K. (1993) The planthormone abscisic acid mediates the drought-inducedexpression but not the seed-speci®c expression of rd22, agene responsive to dehydration stress in Arabidopsis thaliana.Mol. Gen. Genet. 238, 17±25.

Zhou, L., Jang, J.-C., Jones, T.L. and Sheen, J. (1998) Glucose andethylene signal transduction crosstalk revealed by anArabidopsis glucose-insensitive mutant. Proc. Natl Acad. Sci.USA, 95, 10294±10299.



Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene...

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