Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves

Soluble starch synthase I: a major determinant for thesynthesis of amylopectin in Arabidopsis thaliana leaves

David Delvalle1, Sylvain Dumez1, Fabrice Wattebled1, Isaac Roldan2, Veronique Planchot3, Pierre Berbezy4, Paul Colonna3,

Darshna Vyas4, Manash Chatterjee4,†, Steven Ball1, Angel Merida2 and Christophe D’Hulst1,*

1Unite de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS/USTL, IFR 118, Universite des Sciences et Technologies

de Lille, 59655 Villeneuve d’Ascq Cedex, France,2Instituto de Bioquimica Vegetal y Fotosintesis, CSIC-USE Avda Americo Vespucio s/n, Isla de la Cartuja, 41092 Seville, Spain,3Unite de Recherche sur les Polysaccharides leurs Interactions et leurs Organisations, Centre INRA de Nantes, rue de la

Geraudiere, B.P. 71627, 44316 Nantes Cedex 3, France, and4Biogemma UK Ltd, 200 Science Park, Milton Road, Cambridge CB4 0GZ, UK

Received 29 March 2005; accepted 6 May 2005.*For correspondence (fax þ33 3 20 43 65 55; e-mail [email protected]).†Present address: National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK.

Summary

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants.

These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant

kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to

have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes

has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform

belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin.

Comparative analysis of b-limit dextrins for both wild type and mutant amylopectins suggests a specific and

crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own

characterization of SSI activity and the previously described kinetic properties of maize SSI, our results

suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin

cluster synthesis.

Keywords: Arabidopsis, starch, amylopectin structure, starch synthase, a-glucan.

Introduction

Starch accumulates in plants as a highly ordered mixture of

amylopectin and amylose. Both macromolecules are solely

made of glucose residues that are linked together by a-1,4

(linear chains) and a-1,6 (branch points) O-glycosidic bonds

(Buleon et al., 1998). Structural organization and physico-

chemical properties of amylopectin and amylose are clearly

distinct. The branching level is mostly responsible for these

differences. Amylopectin is the major component of starch

(up to 80% of starch weight) and is a highly branched

molecule (5–6% of a-1,6 linkages) that adopts a cluster-like

organization (Hizukuri, 1986; Manners, 1989). Conversely,

amylose is an infrequently branched molecule (<1%) whose

organization within the granule remains obscure.

Metabolism of starch is under the control of a large panel

of enzymatic activities commonly found in several distinct

isoforms (Ball and Morell, 2003; Myers et al., 2000). This

multiplicity of enzyme forms makes it difficult to understand

the specific role of each component in the pathway. The

presence of multiple starch synthases is an ancient trait

correlating with the presence of starch metabolism within

the plastid of all green plants. This was recently demonstra-

ted by the finding of six sequences coding the same classes

of enzymes in the genomes of those green algae that are

thought to have diverged at the earliest stage from the

common ancestor of the green lineage (Ral et al., 2004).

Starch synthases (EC 2.4.1.21) elongate glucans by addition

of a glucose residue from ADP-glucose to their non-reducing

ends (NREs) through the formation of a-1,4 linkages. One

form of starch synthase, granule bound starch synthase I

(GBSSI) is specifically associated with the starch granule

398 ª 2005 Blackwell Publishing Ltd

The Plant Journal (2005) 43, 398–412 doi: 10.1111/j.1365-313X.2005.02462.x

where it is responsible for the synthesis of very long glucans

such as those found in amylose and some subfractions of

amylopectin (Delrue et al., 1992; Maddelein et al., 1994). The

precise molecular mechanism by which this enzyme syn-

thesizes amylose is still under debate (Ball et al., 1998;

Denyer et al., 1996; van de Wal et al., 1998; Wattebled et al.,

2002). However, the analyses of numerous plant mutants

have shown that GBSSI activity is required for amylose

synthesis (Delrue et al., 1992; Denyer et al., 1995; Hovenk-

amp-Hermelink et al., 1987; Tsai, 1974). Other forms of

starch synthases belonging to a minimum of four sequence

families have been detected in the genomes of various

species ranging from the picophytoplanktonic green alga

Ostreococcus tauri (Ral et al., 2004) to rice (Baba et al., 1993;

Tanaka et al., 1995). Because of their conservation through-

out the plant kingdom, it is reasonable to assume that they

play specific and conserved functions in polysaccharide

metabolism. Some of them such as SSI and SSII may also

be, in part, associated with the starch granule in planta

(Denyer et al., 1993; Edwards et al., 1996; Mu-Forster et al.,

1996). However, the amount of activity trapped within the

granule seems to be highly variable between species.

Soluble forms have been classified according to their

sequence homology in five different groups (Li et al., 2003)

named SSI, II, III, IV and V. To date, mutants or antisense

plants (with observable phenotype) have only been des-

cribed for the SSII and SSIII forms (Abel et al., 1996; Fontaine

et al., 1993; Gao et al., 1998; Marshall et al., 1996; Morell

et al., 2003; Zhang et al., 2004). In both cases, mutations lead

to a structural modification of amylopectin associated with

an increase in amylose content. In some cases, multiple

mutants or transgenic lines have been produced (Edwards

et al., 1999; Jobling et al., 2002; Lloyd et al., 1999; Maddelein

et al., 1994). Mutation combinations involving GBSSI, SSII

and SSIII defects always lead to a profoundly modified

amylopectin structure and a strong modification of starch

properties. However, these experiments have not yet led to a

clear understanding of the function of the soluble starch

synthases in starch granule biogenesis. This is mainly due to

the absence of characterization of mutants defective in the

other three classes of soluble starch synthase activities.

Kossmann et al. (1999) described antisense inhibition of SSI

in potato. Although SSI activity in potato tubers was

repressed to non-detectable levels (zymograms), neither

amylopectin synthesis nor structure was affected in the

tubers of the transgenic lines that were considered. The

absence of phenotype was explained by the low expression

level of SSI mRNA in tubers compared with that in leaves. In

one particular review a mutant of SSI was reported to modify

the structure of rice endosperm amylopectin. However, in

that case, the modification was subtle and the description of

the phenotype incomplete (Nakamura, 2002). As a result, the

level of implication of these enzymes in amylopectin synthe-

sis is difficult to assess, although extensive biochemical

studies have been carried out on the SSI isoform from maize

(Boyer and Preiss, 1979; Commuri and Keeling, 2001; Mu

et al., 1994).

To understand the in vivo function of SSI we selected

mutant lines of Arabidopsis thaliana that are selectively

defective for this activity. We demonstrate that SSI is a

plastidial enzyme and that its presence in the leaves of

Arabidopsis is critical for the synthesis of normal amylo-

pectin. We show that the absence of SSI yields a novel

type of amylopectin structure. Kinetic parameters of the

recombinant SSI was measured after expression in

Escherichia coli indicating that Arabidopsis SSI is bio-

chemically related to maize SSI. The function of SSI in the

leaves of Arabidopsis during amylopectin synthesis is

discussed in the light of its biochemical properties and the

consequence of its absence on the structure of amylopec-

tin in the mutant lines.

Results

Selection of the mutant lines defective in SSI

Five loci corresponding to starch synthases have been

detected in the genome of A. thaliana: AtGBS1 (At1g32900),

AtSS1 (At5g24300), AtSS2 (At3g01180), AtSS3 (At1g11720)

and AtSS4 (At4g18240). These loci are related to GBSSI, SSI,

SSII, SSIII and SSIV type of enzymes respectively (according

to http://www.starchmetnet.org/GeneList/GeneListFrameset.

htm). A sixth locus (At5g65685), possibly related to the

starch synthase family, was detected in the genome of this

plant. Although this sequence appears to be related to starch

synthases, it lacks one of the highly conserved regions found

in all starch synthase enzymes (this gene therefore may not

encode an active soluble starch synthase). The other loci

define five distinct classes of starch synthases (Li et al.,

2003). One class represents GBSSI, whereas the other clas-

ses correspond to soluble starch synthases putatively

involved in amylopectin synthesis. Gene AtSS1 is located on

chromosome V and is composed of 15 exons and 14 introns

encoding a protein of 652 amino acid with a predicted mass

of 72 081 Da and shows a high level of similarity with SSI

from other plant sources (from 55% identity with maize SSI

to 64% identity with potato SSI). Bioinformatic analysis

predicted the presence of a chloroplast-targeting signal

consisting of the first 49 amino acids giving a 67-kDa mature

protein (ChloroP at http://www.cbs.dtu.dk/services/ChloroP/;

Emanuelsson et al., 1999).

Two independent mutant alleles named Atss1-1 and

Atss1-2 corresponding to T-DNA insertions at gene AtSS1

were detected in the mutant collections generated at URGV

(Versailles, France; http://flagdb-genoplante-info.infobio-

gen.fr/projects/fst/, Balzergue et al., 2001) and Syngenta

(SAIL collection: http://www.syngentabiotech.com/; Ses-

sions et al., 2002) respectively. Single homozygous mutant

SSI mutant of Arabidopsis 399

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

plants were selected and were analysed in detail at the

molecular level and for starch accumulation.

In both mutant lines, the T-DNA insertion is located in the

first intron at position þ392 and þ337 from the start codon

for Atss1-1 and Atss1-2 respectively (Figure 1). Segregation

values observed for kanamycin (URGV line) and Basta

(Syngenta line) resistance (three-fourth resistant and one-

fourth sensitive individuals after self-pollination of a hetero-

zygous individual) indicate that both lines contain only one

insertion of the T-DNA in their nuclear genomes. RT-PCR

experiments showed that a modified messenger corres-

ponding to gene AtSS1 is still expressed in Atss1-1 and

Atss1-2. However, it is unlikely that this transcript could be

translated to give an active protein as its structure is

modified as shown in Figure 1. Indeed no amplification

was obtained when RT-PCR experiments were performed on

a region spanning the insertion site of the T-DNA. Hybrid

fragments containing both T-DNA and AtSS1 were amplified

when RT-PCRs were performed with AtSS1 and T-DNA-

specific primers (data not shown). Intron 1 is either not

correctly removed during the splicing process or the

synthesis of the downstream sequence of AtSS1 (down-

stream of the T-DNA insertion site) is initiated within the T-

DNA itself giving a hybrid transcript.

To test whether a protein corresponding to SSI is still

present or not in the mutant lines, Western blot analysis was

carried out with a rabbit antiserum raised again the peptide

‘GTGGLRDTVENC’ corresponding to a highly conserved

region located at the C-terminal end of all known starch

synthases. This antibody has been previously shown to

specifically recognize starch synthases from both potato and

Chlamydomonas (Abel et al., 1996; Buleon et al., 1997). A

very similar sequence can be found in SSI protein

(GTGGLRDTVENF). A band with a mass of approximately

67 kDa (the size of the mature protein) is clearly missing in

the leaves of both Atss1-1 and Atss1-2 mutant lines

(Figure 2c) but is still present in Atss3-1 and Atss4-1 mutant

lines defective in SSIII and SSIV types of starch synthase

respectively (Figure 2c) and in the Atss2-1 mutant line

defective in SSII (data not shown).

Enzymological characterization of the Atss1 mutant lines

Soluble starch synthase activities were tested both by zym-

ograms and in vitro assays in wild type and mutant lines. In

the leaves of the wild-type ecotype, two main starch syn-

thase activities were detected by zymograms (Figure 2a,b).

These two activities are dependent on the addition of ADP-

glucose in the incubation buffer (data not shown). One

activity shows a very low mobility and remains situated at

the edge of the stacking gel. The second activity displays a

higher electrophoretic mobility, which may indicate a lower

affinity of the enzyme for glycogen. The latter is missing in

both Atss1-1 and Atss1-2 mutant lines, while the former is

lacking in the mutant lines of Arabidopsis affected at gene

AtSS3 (locus At1g11720; data not shown). These two bands

have remained unaffected by mutations at genes AtSS2

(At3g01180) and AtSS4 (At4g18240) (data not shown).

Therefore, these low and high migrating bands have been

named SSIII and SSI respectively.

No other soluble starch synthase activities can be detec-

ted by this zymogram technique (even when highly sensitive

zymograms were performed with 14C-labelled ADP-glucose,

data not shown). Serial dilutions of the extract were

performed to assess the detection limit of the highly

sensitive radiolabelled zymograms. These were detectable

down to 0.1% of the undiluted SSI activity. Two main

reasons can explain the absence of other starch synthase

activities on glycogen-containing zymograms: (i) other sol-

uble starch synthases may not be expressed in the leaves of

Arabidopsis or expressed at such low level that their

detection is not possible with this method; (ii) glycogen

Figure 1. Intron–exon organization of gene AtSS-1 (gDNA) and structure of

the corresponding mRNA. Insertion sites are indicated as small flags within

the first intron of the gene (precisely at position þ392 from the start codon for

Atss1-1 and þ337 for Atss1-2). RT-PCR amplifications of the SSI transcript

were carried out on wild type and mutant lines. Three targeted regions were

amplified: one upstream, one spanning and one downstream of the insertion

site. The results of RT-PCR amplification (2% agarose gel stained with

ethidium bromide) are shown for both Atss1-1 and Atss1-2 mutant alleles

compared with the corresponding wild-type ecotype.

400 David Delvalle et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

might not be used as an efficient primer by these soluble

starch synthases. A last explanation is that SSII and SSIV

functions might not be restricted to glucan elongation in the

leaves of Arabidopsis. Further work is required to answer

this question.

Soluble starch synthase activity was determined by

in vitro assays with radio-labelled ADP-glucose (14C). Total

SS activity is decreased by approximately 65% in Atss1-1

and 72% in Atss1-2 lines (Table 1). This result correlates the

observation performed on glycogen-containing zymograms

that show the absence of one of the two major soluble starch

synthase activities in both mutant lines.

A chloroplast-enriched leaf extract was produced in order

to determine intracellular localization of soluble starch

synthases (these activities are detectable on a zymogram).

To test the purity of the chloroplast-enriched extract, plastid

integrity was checked using optical microscopy (Supple-

mentary Material). Furthermore, starch phosphorylase and

b-amylase activities was tested on zymograms (intracellular

localization of these enzymes has already been described in

other papers: Zeeman et al., 1998, 2004) (Supplementary

Material). Our results indicate that both SSIII and SSI

activities are localized within chloroplasts and are likely to

be active during starch metabolism in Arabidopsis leaves.

Other starch-metabolizing enzymes remain unaffected by

a mutation at the AtSS1 locus. This was shown by both

zymograms and in vitro enzyme assays for hydrolases,

branching enzymes, pullulanases, starch-phosphorylases,

phosphoglucomutase and ADP-glucose pyrophosphorylase

(Figure 3; Table 1). Moreover, no modification of GBSSI

(a) (b)

(c)

Figure 2. (a, b) Zymogram of soluble starch

synthases (SSs) from leaf extract performed

under native conditions. After migration of

150 lg of proteins on a glycogen-containing gel

and incubation overnight at room temperature

with 1 mM ADP-glucose, SS activities were

revealed by soaking the gel for 30 min in lugol

solution (I2/KI) and washed several times with

distilled water before the picture was taken.

Lanes WS and Col-0: wild-type references cor-

responding to ecotypes Wassilewskija and

Columbia respectively. Lanes Atss1-1 and

Atss1-2: SSI mutant samples.

(c) Western blot analysis of different SS mutants

of Arabidopsis thaliana. Soluble proteins

(100 lg) from a leaf crude extract were loaded

and separated onto an SDS-PAGE gel (7.5%

acrylamide). After transfer onto the PVDF mem-

brane, the blot was probed overnight at 4�C with

antiserum PA55 (dilution 1:200 in TBS). WS and

Col-0, wild-type samples (Wassilewskija and

Columbia ecotypes respectively). Atss1-1 and

Atss1-2, SSI mutant samples. AtSS3 and AtSS4

lanes correspond to leaf extracts of mutant lines

defective in genes At1g11720 and At4g18240

respectively (corresponding to SSIII and SSIV

type of soluble SSs).

Table 1 In vitro assays of several starch-metabolizing enzymes performed with leafcrude extracts of wild type and Atss1-1lines. Activities are expressed in nmolmin)1 mg)1 of proteins � standard error(in each case n ¼ 3) except for a-amylasefor which the activity level is expressed inarbitrary units (‘Ceralpha units’ as des-cribed by the manufacturer)

Measured activity WS Atss1-1

Soluble starch synthase (with rabbit liver glycogen as primer) 2.01 � 0.15 0.89 � 0.14AGPase (without 3-PGA) 18 � 4.5 13 � 1.4AGPase (þ3 mM 3-PGA) 31 � 6.9 36 � 9.9Starch branching enzyme 454 � 44 522 � 31a-amylase (Ceralpha units · 10)4) 3.9 � 0.05 4.2 � 0.07b-amylase 3.4 � 0.5 3.5 � 0.7Maltase 0.47 � 0.06 0.53 � 0.09a-1,4-glucanotransferase 0.32 � 0.04 0.35 � 0.05Pullulanase 615 � 12 642 � 26Starch-phosphorylase 892 � 15 1100 � 41

Col-0 Atss1-2

Soluble starch synthase (with rabbit liver glycogen as primer) 5.20 � 0.14 1.95 � 0.17

SSI mutant of Arabidopsis 401

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

content in starch can be detected between wild type and

mutant lines (Supplementary Material). From these results,

we can conclude that mutations in AtSS1 specifically affect

the activity of SSI in Arabidopsis leaves without significantly

disturbing any other starch-metabolizing enzyme.

To get a better view on the biochemical properties of SSI,

the recombinant enzyme was produced in E. coli. A cDNA

corresponding to the whole coding sequence of SSI was

amplified and introduced in a destination vector (pDEST15)

in order to add a GST tag to the N-terminal end of the

recombinant protein. This allowed its purification by affinity

chromatography performed on a glutathione-substituted

column. After purification of the recombinant protein, the

zymogram technique for soluble starch synthases and

hydrolases was used to test whether SSI is still active and

to select for fractions devoid of any endogenous elongation

and hydrolytic activities (Supplementary Material). The Km

for ADP-glucose measured on glycogen as a primer is

0.075 mM. It is on the same order of magnitude as for maize

SSI (0.1 mM; Cao et al., 2000). The activity of the recombin-

ant SSI was tested on several glucan primers ranging from

maltose to maize amylopectin. The results are summarized

in Table 2. The activity level of recombinant SSI was

expressed relative to the number of available NREs for the

different acceptor molecules used in this test because each

NRE potentially represents a substrate for the enzyme (inner

glucose residues cannot be used as substrate for the

enzyme). While the number of NRE was precisely calculated

for malto-oligosaccharides (DP2–DP7) it has only been

estimated for maize b-limit dextrins (BLD), maize amylopec-

tin and rabbit liver glycogen based on the characteristics

commonly accepted for these molecules (see Table 2 for

(a)

(b)

(d)

(c)

Figure 3. Zymograms of starch-metabolizing

enzymes. Approximately 100 lg of native pro-

teins from a leaf crude extract was loaded onto

the different gels. WT, wild type (WS ecotype);

Atss1-1, mutant line.

(a) Zymogram of starch-modifying activities per-

formed on a starch-containing polyacrylamide

gel. After migration and incubation overnight at

room temperature, starch-modifying enzyme

activities were revealed by iodine staining. Iden-

tification of the different bands was established

following the analysis of several mutated plants

of Arabidopsis defective in each of the specified

activity (data not shown): SBE2.2, starch-branch-

ing enzyme 2.2 (gene At5g03650); SBE2.1, starch-

branching enzyme 2.1 (gene At2g36390);

ISO1, isoamylase (dbe1); PUL, pullulanase

(At5g04360); RAM-1, b-amylase (cytosolic form;

At4g15210).

(b) Zymogram of starch-phosphorylases per-

formed on a glycogen-containing polyacryla-

mide gel. After separation and incubation

overnight at room temperature in the presence

of Glc-1-P at 20 mM, starch-phosphorylase activ-

ities were revealed by iodine staining. cSTP,

cytosolic form of starch-phosphorylase

(At3g46970); pSTP, plastidial form of starch-

phosphorylase (At3g29320).

(c) Zymogram of phosphoglucomutases per-

formed on polyacrylamide gel. After migration,

the gel was incubated at room temperature in a

buffer specific to PGM.

(d) Zymogram of pullulanase performed on an

azure pullulan-containing polyacrylamide gel.

After migration, the gel was incubated overnight

at room temperature. Pullulanase activity ap-

pears as a white band on the gel.

402 David Delvalle et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

more details). SSI activity regularly increases with the size of

the acceptor malto-oligosaccharide, although maltose does

not represent an effective substrate for the recombinant

enzyme. However, branched glucans represent much better

substrates for the enzyme as witnessed by the higher level of

activity measured with BLD when compared with DP2 and

DP3 and glycogen when compared with DP7. The average

outer chain length (OCL) of branched glucans clearly influ-

ences the activity of SSI. While SSI is strongly active on

glycogen (OCL ¼ 7–8), it is less active on maize amylopectin

(OCL ¼ 12–14) and much less active on maize BLD

(OCL ¼ 2.5). The same behaviour has been described for

maize SSI (Commuri and Keeling, 2001) indicating that both

enzymes are closely related and might have the same

function in both plants. The activity of SSI was also tested

on the Arabidopsis amylopectin purified from both the wild

type and the Atss1-1 mutant lines. SSI is almost four times

more active on wild-type amylopectin suggesting that the

structure of mutant amylopectin is modified and not com-

patible with the optimal activity of SSI. These modifications

are likely to concern the most outer chains within amylo-

pectin that are expected to define the best available

substrates for SSI.

Phenotypic characterization of the mutant line

Germination percentages, rates of growth, flowering capa-

cities and formation of siliques were recorded for Atss1-1

and Atss1-2 mutant lines. All these parameters were com-

parable with those of the wild-type reference strain. Starch

levels and structure were also compared with those of the

wild-type reference. Interruption of the AtSS1 locus led to a

near-23% decrease in leaf starch level measured at the end of

the illuminated period (Table 3). However, both the wild

type and the mutant plants were essentially devoid of starch

at the end of the dark period. No accumulation of water-

soluble a-glucans was recorded in this line. The kmax of the

iodine–polysaccharide complex measured for the total

starch (after lipid removal) was slightly increased in the

mutant (from 562 nm in wild type to 567 nm in Atss1-1)

suggesting the presence of a higher level of amylose

(Table 3). Size exclusion chromatography performed on

sepharose CL-2B columns (Figure 4) and subsequent amy-

loglucosidase assay of the fractionated glucans demon-

strated that the amylose content increased from 19% of the

total starch in the wild-type line to 31% in the mutant

Table 2 Determination of the glucan primer preference of therecombinant SSI expressed in Escherichia coli. Results are themean of three independent assays (� SE) and are expressed innmol min)1 mg)1 of proteins

Glucan primeraMeasuredactivity

Relativeactivityb

Maltose (DP2) 0.09 � 0.03 0.05Maltotriose (DP3) 4.01 � 0.18 3.35Maltopentaose (DP5) 13.4 � 0.87 18.4Maltohexaose (DP6) 12.2 � 0.74 20.0Maltoheptaose (DP7) 20.6 � 1.19 39.4Maize b-limit dextrins (OCL ¼ 2.5) 26.0 � 0.80 26.2Maize amylopectin (OCL ¼ 12–14) 26.6 � 1.86 59.6Rabbit liver glycogen (OCL ¼ 7–8) 60.3 � 1.46 180Atss1-1 amylopectinc 9.90 � 0.54 ØWild-type amylopectinc 36.2 � 1.42 Ø

OCL, average outer chain length; Ø, not applicable.aThe final concentration of the glucan was 10 mg ml)1 except foramylopectin purified from the wild type and the mutant (Atss1-1)Arabidopsis lines: 1 mg ml)1.bThe activity measured for SSI has been expressed to the estimatednumber of non-reducing ends (NREs) available to the enzyme duringthe reaction for each substrate (nmol min)1 mg)1 10)18 NREs). Formalto-oligosaccharides (DP2–DP7) the number of NREs that isactually equivalent to the concentration of the molecule within thereaction buffer has been precisely calculated. However, this value hasonly been estimated for amylopectin, b-limit dextrins (BLD) andglycogen. The number of NREs for amylopectin and BLD wasestimated taking into consideration the following parameters: 105

glucose residues per amylopectin molecule with an average branch-ing level of 6%. Amylopectin b-amylolysis limit ¼ 55% (i.e. 45% ofremaining material after the digestion of amylopectin by b-amylase).For glycogen: 5.5 · 104 glucose residues per molecule with anaverage branching level of 9%.cAmylopectin was purified by size exclusion chromatography onsepharose CL-2B column.

Table 3 Leaf starch accumulation in wild type (ecotype WS) and Atss1-1 lines. The results presented in this table are the average of fiveindependent experiments (mean � SE). Starch was extracted at the end of the illuminated period. Amounts of starch, amylose and WSP weredetermined by amyloglucosidase assays (see Experimental procedures). The kmax of amylopectin and the amount of amylose were measuredafter purification through size exclusion chromatography on sepharose CL-2B columns

Wild type (WS) Atss1-1 P-valuea

Starch content (mg g)1 of FW) (n ¼ 5) 2.18 � 0.2 1.68 � 0.19 0.0384kmax of total starch (n ¼ 8) 562 � 2 nm 567 � 2 nm 0.0801kmax of amylopectin (n ¼ 8) 558 � 1 nm 564 � 3 nm 0.0773Proportion of amylose (n ¼ 6) 18.6 � 2.5% 30.6 � 2.4% 0.0036Total WSP content (mg g)1 of FW) Not detected Not detected Ø

kmax, wavelength at the maximal absorbance of the iodine–polysaccharide complex; FW, fresh weight; WSP, water-soluble polysaccharides.aCalculation based on Student’s two-sample t-test; Ø, not applicable.

SSI mutant of Arabidopsis 403

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

(Table 3). Amylopectin accumulation was decreased by

about 33% in the mutant line. However, if one expresses

amylose content relative to the amount of leaf material there

is no significant increase in this particular polysaccharide

fraction. Thus, the increase in the amylose-to-amylopectin

ratio (0.26 in wild type to 0.45 in mutant) occurs simply

because of reduced amylopectin synthesis and not because

of a substantial increase in amylose synthesis.

The kmax of the amylopectin–iodine interaction is only

slightly increased in the starch of the Atss1-1 and Atss1-2

mutant lines (Table 3; Figure 4). To determine whether

structural modifications occur in mutant amylopectin, this

polymer from both mutant sources was subjected to com-

plete enzymatic debranching. The chain length (CL) distri-

bution was then determined by FACE after coupling with

APTS and/or by high performance anion exchange chroma-

tography with pulsed amperometric detection (HPAEC-PAD)

(Figure 5). Similar results were obtained with both tech-

niques. The wild-type amylopectin showed a polymodal

distribution of CL with a maximum at DP ¼ 11, 12

(Figure 5a,d). This type of CL distribution for amylopectin

is commonly observed throughout the plant kingdom.

However, the CL distribution is profoundly modified in the

mutant amylopectin and has become unimodal (although a

small secondary peak might be observed at DP ¼ 7; Fig-

ure 5b,e). DP ¼ 13, 14 and 15 are the most abundant glucans

in the amylopectin of the mutant while chains of DP ¼ 8 to

12 are dramatically reduced (Figure 5c,f). Conversely, chains

of DP ¼ 17–20 are significantly increased in the mutant

amylopectin as shown in Figure 5(c,f). This kind of CL

distribution has never been recorded in plants.

The amylopectin structure for both WS and Atss1-1

mutant was analysed after b-amylolysis. b-amylase is an

exoenzyme that releases maltose from the NRE of a glucan

after the cleavage of a-1,4 linkages. However, this enzyme is

unable to digest the branches and stops two to three

residues from the branch points (a-1-6 bonds). Therefore,

the outer chains of amylopectin are almost completely

degraded after b-amylolysis while internal chains are degra-

ded to the most outer branch point leading to the production

of so-called BLD. CL distributions of the corresponding BLD

was established by HPAEC-PAD and are presented in

Figure 6(c,d) for wild type and mutant amylopectin respec-

tively. The differential profiles corresponding to wild type

and mutant amylopectin are shown in Figure 6(e,f) respec-

tively. A distinct excess of DP < 10 chains can be observed in

both lines. On the contrary, the number of chains of up to 30

glucose residues are reduced, with the notable exception of

DP ¼ 17, 18 and 19 in the wild-type amylopectin. This result

was reproduced in two independent experiments indicating

that the excess of DP ¼ 17, 18 and 19 chains is not an artefact

but rather represents a specific feature that discriminates b-

amylolysis susceptibility between wild type and mutant

amylopectins. CL distributions of wild type and mutant BLD

are different as witnessed by the differential profile in

Figure 6(g) that shows more chains of DP < 10 and less

chains of 11 < DP < 24 in the mutant polysaccharide.

The branching level of amylopectin, measured by

methylation analysis, remained unaffected in Atss1-1

(8.0 � 0.3% in wild type and 7.0 � 1.3% in Atss1-1; these

Figure 4. Comparison of size exclusion chromatography profiles of total

starch from wild type and Atss1 mutant lines. Starch (1.5 mg) dissolved in

NaOH (10 mM) was loaded on a sepharose CL-2B column. Fractions of 300 ll

were collected at a flow rate of 12 ml h)1. (a) WS. (b) Atss1-1. (c) Col-0.

(d) Atss1-2. The y-axis indicates the maximal absorbance of the iodine–

polysaccharide complex measured for each fraction. The x-axis corresponds

to the elution volume in ml. Numbers indicate the kmax of amylopectin in nm.

404 David Delvalle et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

values were calculated from two independent experi-

ments).

The impact of the mutation on both the crystallinity and

the morphology of starch granules was studied by both wide

angle X-ray scattering and transmission electron microsco-

py (TEM). Despite the strong modification of the amylopec-

tin structure and the increase in the amylase-to-amylopectin

ratio in the mutant starch, crystallinity levels and allomorph

configurations were basically the same (both wild type and

mutant starches display a B-type crystallinity whose level is

of 37 � 5%). TEM observations of ultra thin slices of starch

granules and subsequent image analysis have shown that

the granule shape becomes slightly more elongated (Fig-

ure 7a,b). This feature has already been described for

amylose-enriched starches from maize (Duprat et al.,

1980). Nevertheless, no strong modification of the starch

granule morphology was observed. Image analysis was

performed on 1000 granules. Each sample indicates that the

average surface of the mutant granules is lower than that of

the wild-type granules. Figure 7(c) is a differential diagram

that represents the difference in the number of granules

between wild type and mutant lines for several classes of

granule surfaces expressed in lm2. This diagram indicates

that, on average, there are more small starch granules in the

mutant than in the wild-type line.

Expression pattern of SSI

The expression of SSI in the plant was tested by semi-

quantitative RT-PCR. This experiment was performed with

(a) (d)

(e)(b)

(f)(c)

012345678

4 7 10 13 16 19 22 25 28 31 34 37 40012345678

4 7 10 13 16 19 22 25 28 31 34 37 40

012345678

4 7 10 13 16 19 22 25 28 31 34 37 40012345678

4 7 10 13 16 19 22 25 28 31 34 37 40

–2

–1

0

1

2

3

–2

–1

0

1

2

3

4 7 10 13 16 19 22 25 28 31 34 37 40 4 7 10 13 16 19 22 25 28 31 34 37 40

Degree of polymerization

Rel

ativ

e p

rop

ort

ion

of

each

DP

in %

Figure 5. Comparison of the chain length (CL) distribution profiles for wild type and mutant amylopectins. (a) WS. (b) Atss1-1. (d) Col-0. (e) Atss1-2. After

purification on CL-2B column, amylopectin was debranched with isoamylase and pullulanase. The resulting glucans were analysed by HPAEC-PAD. The relative

proportions for each glucan in the total population are expressed as a percentage of the total number of chains (thin vertical bars correspond to standard deviation

calculated for each DP; the presented results are the average of two independent experiments). (c, f) Difference plots between the wild type and the mutant lines

(c ¼ b ) a; f ¼ e ) d).

SSI mutant of Arabidopsis 405

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

several tissues (leaves, flowers and stems). Our results indi-

cate that SSI is expressed at similar levels throughout the

plant (Supplementary Material). This result closely correlates

with that of MPSS data generated for Arabidopsis (available

at http://mpss.udel.edu/at/java.html). Moreover, the expres-

sion pattern of SSI in the leaves was established by semi-

quantitative RT-PCR through a 12 h day/12 h night cycle

(Supplementary Material). It was found that the level of

mRNA is at maximum levels at the end of the illuminated

period and progressively decreases during the night down to

a minimum level 3 h before lights are turned on (three inde-

pendent experiments). These results closely match those

described for transcriptomics experiments in short- and

long-day periods (available at http://www.starchmetnet.org/

Datapages/AtSS1/AtSS1Frameset.htm). This suggests that

the increase in SSI mRNA level anticipates the onset of light.

Nevertheless, the observed oscillation at the mRNA level

correlated neither with fluctuations in protein content

(Western blot) nor with those of enzyme activity levels

(zymogram) (Supplementary Material). Therefore, SSI

seems to be regulated by some additional unidentified post-

transcriptional mechanisms in Arabidopsis leaves.

Discussion

This work reports the phenotypic analysis of two inde-

pendent mutant lines of A. thaliana having a T-DNA insertion

in the AtSS1 (At5g24300) gene encoding a type I form of

starch synthase. These insertion mutations lead to the syn-

thesis of abnormal transcripts together with the absence of a

protein of 67 kDa that matches the predicted mature size for

SSI, which is able to interact with an antibody raised against

one of the highly conserved regions of starch synthases. We

have also shown that in both T-DNA insertion lines, one

soluble starch synthase activity is missing although other

starch-metabolizing enzymes remain unaffected by the

mutation. Therefore, the phenotypic effects observed on

starch metabolism in both lines arise from the deficiency in

SSI activity encoded by the AtSS1 locus and cannot be easily

explained by the presence of unidentified secondary muta-

tions. Moreover, biochemical characterization of recombin-

ant SSI expressed in E. coli has shown that this enzyme is

closely related to maize SSI (Commuri and Keeling, 2001).

Although extensive biochemical work has been carried

out on SSI from different plant sources, its function during

1

345678

4 7 10 13 16 19 22 25 28 31 34 37 40012345678

4 7 10 13 16 19 22 25 28 31 34 37 40

0

1

2

3

4 7 10 13 16 19 22 25 28 31 34 37 40

012345678

4 7 10 13 16 19 22 25 28 31 34 37 40012345678

4 7 10 13 16 19 22 25 28 31 34 37 40

0123456

4 7 10 13 16 19 22 25 28 31 34 37 40

–2

–1

–2

–1

–2–3

–1

0

1

2

3

4 7 10 13 16 19 22 25 28 31 34 37 40

2

0

(a)

(b)

(c)

(d)

(g)

(f)

(e)

Degree of polymerization

Rel

ativ

e pr

opor

tion

of e

ach

DP

in %

Figure 6. Comparison of the chain length (CL) distribution profiles for wild type and mutant amylopectins after b-amylolysis. (a) WS wild-type amylopectin.

(b) Atss1-1 amylopectin. (c) Wild-type amylopectin after b-amylolysis. (d) Atss1-1 amylopectin after b-amylolysis. After purification on CL-2B column, amylopectin

was subjected to b-amylolysis prior to debranching with isoamylase and pullulanase. The resulting glucans were analysed by HPAEC-PAD. The relative proportion

for each glucan is expressed as a percentage of the total number of chains (thin vertical bars correspond to standard variation calculated for each DP; the presented

results are the average of two independent experiments). (e, f) Difference plots between the CL distribution profiles before and after b-amylolysis for the wild type

and mutant amylopectin respectively (e ¼ c ) a; f ¼ d ) b). (g) Difference plot between CL distribution profiles of wild type and mutant amylopectins after

b-amylolysis (g ¼ d ) c).

406 David Delvalle et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

starch synthesis process remains obscure, as no mutant or

transgenic plant with a convincing phenotype on starch

metabolism has ever been described in detail (Boyer and

Preiss, 1979; Commuri and Keeling, 2001; Imparl-Radosev-

ich et al., 1998; Knight et al., 1998; Li et al., 1999; Mu et al.,

1994; Tanaka et al., 1995).

Our results indicate that this form of starch synthase is

essential for the in vivo synthesis of normal amylopectin

in the leaves of Arabidopsis. The absence of SSI leads to

a deficiency in the number of the shorter glucans of

amylopectin (Figure 5) generally assumed to define the

so-called A (most outer chains of amylopectin) and B1

chains (inner chains of amylopectin that belong to only

one cluster) (Hizukuri, 1986). These short glucans are

generally restricted to the amylopectin cluster and are not

long enough to tie clusters together. b-amylolysis of wild-

type amylopectin indicates that chains of DP17-19 are

much less susceptible to degradation than the corres-

ponding chains of the mutant amylopectin (Figure 6e,f).

This result strongly suggests that these chains are located

at different places in mutant and wild-type amylopectins.

DP17-19 chains are preferentially situated at the outer face

of the mutant amylopectin. This observation might

explain why recombinant SSI is much less active with

Atss1-1 amylopectin when compared with wild-type amy-

lopectin (Table 2).

The simplest explanation for the structural analysis

reported in Figure 3 is that SSI is involved in the later

phases of amylopectin synthesis for the filling of the cluster

structure. It has been previously shown that pure recombin-

ant maize SSI cannot elongate glucans above DP20 in length

and remains blocked on such substrates (Commuri and

Keeling, 2001). The affinity of SSI increases exponentially

from DP14 to 20 to a point where the catalytic capacity of the

enzyme is significantly reduced. This observation has been

evoked to explain both the accidental trapping of SSI within

the starch granule and the fact that most of the label

incorporated by the maize SSI in vitro is found in chains that

are less than 10 glucose residues in length. Branching

enzymes are unable to use substrates of less than 12 glucose

residues in length (Guan and Preiss, 1993; Takeda et al.,

1993). These results seem to imply that SSI is unlikely to

generate glucans that are long enough to constitute sub-

strates for the branching enzymes and is consistent with a

function confined to the filling of accessible clusters. It is

striking to note that the chains missing in vivo in the

amylopectin structure (this work) are precisely those which

are effectively synthesized by the recombinant enzyme

(b)(a)

(c)

Figure 7. Transmission electron microscopy of

ultra thin slices of starch granules extracted at

the end of the illuminated period from leaves of

wild type (a) and Atss1-1 (b) lines. Bars ¼ 1 lm.

(c) Difference plot of the surface distribution of

starch granules between the wild type and Atss1-

1 (surface is expressed in lm2). The surface of the

granules was determined after image analysis of

the samples (1000 granules were observed for

each line). This diagram indicates that granules

in line Atss1-1 are statistically smaller than their

wild-type counterpart.

SSI mutant of Arabidopsis 407

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

in vitro (Commuri and Keeling, 2001). This result suggests

that SSI activity displays very little (or even no) overlap with

the activities of other enzymes of the starch-synthesizing

machinery, especially other soluble or granule starch

synthases.

From the perspective of activity, SSI can be viewed as a

major filling enzyme with little capacity to extend chains and

generate suitable substrates for the branching enzymes. A

possible explanation for the fact that an increased number of

longer outer chains is witnessed in the mutant is that the

slow release of SSI by its substrate would therefore tend to

inhibit its replacement by another elongation enzyme and

subsequently tend to terminate the synthesis of a cluster.

Competition, for both primer and substrate, between SSI

and enzymes capable of generating very long chains to

initiate a novel cluster (such as SSIII) might constitute an

effective way to regulate the growth and size of the

amylopectin molecule. The absence of SSI would thus

redirect the activity of one (or several) of the remaining

starch synthases (including GBSSI) towards the synthesis of

longer outer chains in the mutant amylopectin. Generation

of double mutant lines defective in SSI and other starch

synthases would help in defining which isoform is respon-

sible for the observed phenotype in the Atss1-1 line.

Various balances of activity between these isoforms in

different tissues would thus lead to different structures that

would be more or less accessible to degradation. One can

speculate that having more external clusters in transitory

starches of leaves will provide best-suited substrates for a

rapid degradation by exoenzymes such as b-amylases that

are known to be effective in plant leaves (Scheidig et al.,

2002). SSI is indeed thought to have less impact on storage

starch synthesis. Indeed, a preliminary report of a rice

mutant defective in SSI activity in the endosperm (unpub-

lished data reported in a review by Nakamura, 2002) showed

a lesser impact on storage starch structure.

Experimental procedures

Materials

ADP [U-14C] glucose, CL-2B sepharose column and Percoll wereobtained from Amersham Biosciences (Orsay, France). ADP-glucose was from Sigma (Lyon, France). Starch assay kit waspurchased from Enzytec (Toulouse, France).

Arabidopsis lines, growth conditions and media

Wild type (Wassilewskija, WS and Columbia, Col-0) and mutantlines of A. thaliana were obtained from the T-DNA mutant collec-tions generated at URGV, INRA, Versailles (Bechtold et al., 1993;Bouchez et al., 1993) and Syngenta (Sessions et al., 2002). Standardprocedures were employed for plant germination and growth. Theplants were grown on peat-based compost (seeds were previouslyincubated at 4�C before sowing) under 8 h dark/16 h light cycle with

temperature ranging from 16�C (during the night) to 21�C during theilluminated period.

RT-PCR amplifications

Approximately 2 g of fresh tissue was harvested in the middle of thelight phase (see culture conditions described above) for total RNAextraction with the Plant RNeasy kit (Qiagen, Courtabouef, France)following the supplier’s instructions. Twenty nanograms of purifiedtotal RNA was used to perform RT-PCR amplifications using the OneStep RT-PCR kit (Qiagen). Three different regions surrounding theT-DNA insertion site were targeted for amplification: upstream,downstream and spanning the T-DNA insertion site. The followingprimer pairs were used: Upstream region (305 bp fragment) prim-ers – SS5for51: 5¢-AACAACCAGACGAAATCAAACA-3¢ and SS5rev5:5¢-AAGCCAAGAACGGAGCCAGAAC-3¢. Spanning region (286 bpfragment) primers – AtSS5for1: 5¢-TTTCCGTCCGATCGCCAGT-CTC-3¢ and AtSS5rev1: 5¢-TACGCCAAAGTCAGCCATTACAA-3¢.Downstream region (554 bp fragment) primers – SS5for32: 5¢-AT-GTTTGTGGTTCTTTGCCGATAG-3¢ and SS5rev32: 5¢-CCACCCGAC-TGCTCCATACC-3¢.

Determination of starch and WSP contents and spectral

properties of the iodine–starch complex

A full account of amyloglucosidase assays and kmax (maximalabsorbance wavelength of the iodine–polysaccharide complex)measures can be found in Delrue et al. (1992). Starch and water-soluble glucan contents in leaves were determined as described inZeeman et al. (1998).

Extraction and purification of starch

Arabidopsis leaves were harvested at the end of the light period.Approximately 10 g of fresh material was homogenized using aTissue Tearor (Biospec Products, Inc., Bartlesville, OK, USA) in30 ml of the following buffer: 100 mM 3-(N-morpholino) propane-sulphonic acid (MOPS), pH 7.2, 5 mM EDTA, 10% (v/v) ethanediol.The homogenate was filtered through two layers of Miracloth andcentrifuged for 15 min at 4�C and 4000 g. The pellet was resus-pended in 30 ml Percoll 90% (v/v) and centrifuged for 40 min at 4�Cand 10 000 g. The starch pellet was washed six times with distilledsterile water (10 min at 4�C and 10 000 g between each wash).Starch was finally stored at 4�C in 20% ethanol.

Separation of starch polysaccharides by size exclusion

chromatography

Starch (1.5–2.0 mg) was dissolved in 500 ll of 10 mM NaOH andsubsequently applied to a Sepharose CL-2B column (0.5 cmi.d. · 65 cm), which was equilibrated and eluted with 10 mM NaOH.Fractions of 300 ll were collected at a rate of one fraction per1.5 min. Glucans in the fractions were detected by their reactionwith iodine and levels of amylopectin and amylose were deter-mined by amyloglucosidase assays.

Chain length distribution of polysaccharides

Amylopectin CL distribution was established by HPAEC-PAD(Dionex, Voisins Le Bretonneux, France) after complete debranching.

408 David Delvalle et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

A complete description of this technique can be found in Fontaineet al. (1993). Debranching procedure is as follows: after purificationon sepharose CL-2B column, 500 lg of amylopectin was dialysedagainst distilled water and subsequently lyophilized. The amylo-pectin pellet was resuspended in 1 ml of 55 mM sodium acetate, pH3.5, and incubated overnight at 42�C with 20 U of isoamylase (Ha-yashibara Biochemical Laboratories, Okayama, Japan) and 1 U ofpullulanase (Sigma). Salts were subsequently removed by runningthe sample through an ‘Extract-clean’ Carbograph column (Alltech,Deerfield, IL, USA).

b-amylase digestion of amylopectin

After purification on sepharose CL-2B column, 500 lg of amylo-pectin was dialysed against distilled water and lyophilized. Theamylopectin pellet was resuspended in 500 ll of 55 mM sodiumacetate, pH 3.5, and incubated for 4 h at 30�C with 17 U of b-amylasefrom sweet potato (Sigma). To ensure complete digestion, sameamount of b-amylase was added to the sample for a further 20 hincubation (production of reducing power is monitored during thereaction using 3,5-dinitrosalicylic acid assay at 540 nm). Enzymaticreaction was stopped by boiling the sample for 5 min. The samplewas then debranched as described above by isoamylase (20 U) andpullulanase (1 U) and the corresponding glucans were analysed byHPAEC-PAD chromatography (Dionex).

Branching level determination

Branching level of amylopectin was measured by methylationanalysis. Amylopectin (200 lg) was dissolved in 300 ll of anhy-drous dimethyl sulphoxide at 80�C for 1 h. Methylation was per-formed with 500 ll iodomethane in the presence of 50 mg NaOH.The sample was sonicated in a water bath for 1 h and left in the darkovernight at room temperature. The reaction was stopped byaddition of chloroform (2 ml), two crystals of sodium thiosulphateand water (2 ml). The chloroform phase was washed six times withwater (2 ml), dried and lyophilized. Another cycle of methylationwas performed to ensure complete methylation of the polysaccha-ride. The sample was suspended in 25 ll of acetone and washydrolysed in trifluoroacetic acid 4 M (1 ml) for 4 h at 100�C.Trifluoroacetic acid was removed by four cycles of distillation withethanol. The sample was dried. Reduction was performed by addi-tion of BD4Na 20 mg ml)1 (500 ll) in ammoniac for 2 h at roomtemperature. The reaction was stopped by addition of acetic acid5%. Boric acid was removed by six rounds of distillation withmethanol. The sample was dried and lyophilized. Peracetylationwas performed by addition of 500 ll acetic anhydride for 4 h at100�C. The sample was dried and suspended in 1.5 ml of chloro-form. The chloroform fraction was washed six times with 2 ml ofwater and was filtrated on anhydrous Na2SO4. Quantitative analysisof the sample was performed by gas chromatography [Varian 3400apparatus equipped with a flame ionization detector and a glassstick evaporator (Varian, Les Ulis, France)] on a capillary column(EC.Tm-1, 30 m · 0.25 mm; Alltech). Column temperature was setfrom 120 to 240�C, with a temperature gradient of 2�C min)1.Detector temperature was set at 250�C.

Zymogram techniques

Soluble starch synthase activities. Proteins (100 lg) from a leafcrude extract were loaded on a native PAGE (7.5% acrylamide)containing 0.3% of rabbit liver glycogen. After migration (under

native condition for 3 h at 4�C at 15 V cm)1), the gel was incubatedovernight at room temperature in the following buffer: Glygly/NaOH50 mM (pH 9), (NH4)2SO4 100 mM, b-mercaptoethanol 5 mM, MgCl25 mM, BSA 0.25 g l)1 and ADP-Glc 1 mM. Starch synthase activitieswere revealed by iodine solution.

Starch-modifying activities (hydrolases, branching

enzymes). Proteins (100 lg) from a leaf crude extract were loa-ded on native PAGE (7.5% acrylamide) containing soluble potatostarch (0.3% final concentration). After migration (under nativecondition for 3 h at 4�C at 15 V cm)1), the gel was incubated over-night at room temperature in the following buffer: Tris/HCl 100 mM

(pH 7.0), MgCl2 1 mM, CaCl2 1 mM and DTT 1 mM. Starch-modifyingactivities were revealed by iodine staining.

Starch phosphorylases. Proteins (100 lg) from a leaf crudeextract were loaded on native PAGE (7.5% acrylamide) containing0.75% (w/v final concentration) of rabbit liver glycogen and separ-ated for 3 h at 4�C at 15 V cm)1 under native conditions. Aftermigration, the gel was incubated overnight at room temperature insodium citrate 100 mM (pH 7.0) and G-1-P 20 mM (glucose-1-phos-phate). Iodine staining of the gel revealed starch phosphorylaseactivities.

Phosphoglucomutases. Proteins (100 lg) from a leaf crude ex-tract were loaded on native PAGE (7.5% acrylamide). After migra-tion (under native condition for 3 h at 4�C at 15 V cm)1), the gel wasincubated at room temperature in the following buffer: Tris/HCl200 mM (pH 7.0), EDTA 1 mM, MgCl2 50 mM, glucose-1-phosphate15 mM, NAD 0.5 mM, NADP 0.25 mM, glucose-1,6-diphosphate0.05 mM, 17 U of glucose-6-phosphate dehydrogenase, 3-(4,5-di-methylthiazol)-2,5 diphenyltetrazolium bromide 1 mM and 5-methyl-phena-zinium methyl sulphate 0.5 mM. The reaction wasusually stopped after 1 h of incubation at room temperature.

Pullulanase. Proteins (100 lg) from a leaf crude extract wereloaded on native PAGE (7.5% acrylamide) containing azure-pullulan(0.3% final concentration). After migration (under native conditionfor 3 h at 4�C at 15 V cm)1), the gel was incubated overnight at roomtemperature in the following buffer: Tris/HCl 100 mM (pH 7.0), MgCl21 mM, CaCl2 1 mM and DTT 1 mM. Pullulanase activity appears as awhite band on the gel.

In vitro assays of starch-metabolizing enzymes

Soluble starch synthases. Proteins (50 lg) were incubated at30�C for 30 min in 100 ll of the following buffer: Glygly/NaOH50 mM (pH 9), (NH4)2SO4 100 mM, b-mercaptoethanol 5 mM, MgCl25 mM, BSA 0.25 g l)1, [U14C]ADP-glucose (333 mCi mM

)1) 0.75 nM

and glycogen 10 mg ml)1. For the determination of the Km for ADP-glucose, various concentrations of non-radiolabelled ADP-glucosewere added to the buffer. For regular soluble starch synthase assay,1 mM of ADP-glucose was added to the reaction mixture. Thereaction was stopped by boiling for 10 min. After the addition of1 ml of 75% methanol/1% (w/v) KCl solution, the sample was cen-trifuged at 10 000 g at 4�C for 10 min. The pellet was rinsed twicewith 1 ml of 75% methanol/1% (w/v) KCl solution and dried at roomtemperature for 30 min. The pellet was resuspended in 300 ll ofdistilled water, and finally counted in a scintillation counter after theaddition of 2 ml of counting solution (UltimaGold; Perkin-Elmer,Boston, MA, USA).

SSI mutant of Arabidopsis 409

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 398–412

Determination of the kinetics parameters of the recombinant

SSI. An equivalent of 5 lg of proteins was incubated at 30�C for30 min in 100 ll of the following buffer: Glygly/NaOH 50 mM (pH9), (NH4)2SO4 100 mM, b-mercaptoethanol 5 mM, MgCl2 5 mM,BSA 0.25 g l)1, [U14C]ADP-glucose (333 mCi mM

)1) 0.75 nM.10 mg ml)1 of malto-oligosaccharides (DP2–DP7), maize amylo-pectin, maize BLD or rabbit liver glycogen were added to thebuffer depending on the substrate to be tested. A final concen-tration of 1 mg ml)1 was used for incubation with wild type andAtss1-1 mutant amylopectin. Except for malto-oligosaccharides(DP2–DP7) the reaction was stopped by boiling for 10 min. Afterthe addition of 1 ml of 75% methanol/1% (w/v) KCl solution, thesample was centrifuged at 10 000 g at 4�C for 10 min. The pelletwas rinsed twice with 1 ml of 75% methanol/1% (w/v) KClsolution and dried at room temperature for 30 min. The pelletwas resuspended in 300 ll of distilled water, and finally countedin a scintillation counter after the addition of 2 ml of countingsolution.

When incubated with malto-oligosaccharides, the reaction wasstopped by boiling for 10 min and the glucans were elongated byincubation overnight at 30�C with 7.5 U of phosphorylase ‘a’ fromrabbit muscle (Sigma) in the presence of 50 mM of Glc-1-P (finalconcentration). The reaction was stopped by addition of 3.5 ml of75% methanol/1% (w/v) KCl solution and the samples were furthertreated as described above before counting.

ADP-glucose pyrophosphorylase. ADP-glucose pyrophospho-rylase (AGPase) activity was measured upon the degradation ofADP-glucose in the presence of PPi to produce ATP and Glc-1-P. Glc-1-P is transformed to Glc-6-P (glucose-6-phosphate) by phospho-glucomutase. Glc-6-P is oxidized to D-gluconate-6-phosphate byglucose-6-phosphate dehydrogenase and NADP. The production ofNADPH, Hþ is finally monitored at 365 nm.

Proteins (100 lg) from a leaf crude extract were added to areaction mixture to provide a final volume of 500 ll with thefollowing concentration: GlyGly/NaOH 80 mM (pH 7.5), PPi 1 mM,MgCl2 60 mM, glucose-1,6-diphosphate 0.05 mM, NADP 0.25 mM,NAF 10 mM, ADP-glucose 1 mM and acid 3-phosphoglyceric (3-PGA) 3 mM (added when necessary). After 30 min incubation at30�C, the sample was boiled for 10 min to stop the reaction. Thesample was cleared by centrifugation and the supernatant wascollected to measure the concentration of Glc-1-P. After addition of1 U phosphoglucomutase and 1 U glucose-6-phosphate dehydrog-enase, the absorbance was monitored at 365 nm.

a and b-Amylases, pullulanase, a-1,4 glucanotransferase, starch-phosphorylase, starch branching enzymes and maltase activitieswere determined as described in Zeeman et al. (1998).

Western blot analysis

Soluble proteins (100 lg) were separated by electrophoresis onSDS-PAGE gel (7.5% acrylamide and 0.1% SDS). Before blottingproteins onto PVDF membrane (Amersham Pharmacia Biotech),gels were incubated for 10 min in a Western blot buffer [48 mM Tris,39 mM glycine, 0.0375% (w/v) SDS, and 20% methanol]. The transferwas carried out using the Mini Trans-Blot Cell (Bio-Rad, Hercules,CA, USA), for 90 min at 100 mA with the same Western blot buffer.After blocking for 1 h in a 1% milk solution made in TBS-T buffer[20 mM Tris base, 137 mM NaCl, 0.05% Tween20, pH 7.6 with 1 M

HCl], membranes were incubated overnight at 4�C with the anti-serum diluted in TBS buffer [20 mM Tris base, 137 mM NaCl, pH 7.6with 1 M HCl]. After incubation, membranes were rinsed severaltimes in TBS-T buffer at room temperature before immunodetection

with a biotin and streptavidin/alkalin phosphatase kit (Sigma) fol-lowing the supplier’s instruction.

Cloning of the SSI cDNA and expression in E. coli

The open reading frame of SSI was amplified by PCR usingprimers containing the recombination sequences of the GatewayCloning System (Invitrogen, Cergy-Pontoise, France). The PCRproduct was cloned by recombination in the Gateway pDONR201vector following the manufacturer’s instruction. The SSI cDNAwas then inserted by a second recombination step in the GatewaypDEST15 vector, allowing the expression of the N-terminus GST-tagged fusion protein in E. coli BL21 strain. After a 4-h inductionin LB medium containing 0.2% L-arabinose, the GST-tagged pro-tein was purified using the MagneGST purification system fol-lowing the supplier’s instruction.

Transmission electron microscopy

To avoid material losses as well as heterogeneous distribution, wetsamples were first embedded in 3% (w/v) agar solution (tempera-ture below 40�C). After the agar solution had solidified, small cubesof samples (1 mm3) were cut out and treated for 20 min in 1%periodic acid, washed in deionized water, treated in a saturatedsolution of thiosemicarbazide for 24 h, washed again and finallytreated with 1% (w/v) AgNO3 for 24 h (PATAg treatment) accordingto Gallant et al. (1973). Samples were then washed and embeddedin Nanoplast (Helbert et al., 1996). After Nanoplast polymerization(10 days), the samples were again embedded in LR White HardGrade. Sections (0.1 mm thick) were obtained using a diamondknife (Microm MT-7000, Bal-Tec RMC; Tucson, AZ, USA), andexamined under 80 keV with a JEOL (Croissy sur seine, France) 100Stransmission electron microscope.

X-ray diffraction measurements

Crystallinity level of the starch samples was determined by X-raydiffraction as already described in Pohu et al. (2004).

Acknowledgements

This work was supported by Genoplante (program no. Af2001030),the CNRS, the Region Nord-Pas de Calais (program ARCir), theMinistere Delegue a la Recherche (grant no. JC5145 ACI Jeunes-Chercheurs) and the Spanish Ministerio de Educacion y Ciencias(grant no. BIO2003-00431). We are grateful to Frederic Chirat for hisassistance with FACE and Yves Leroy for his assistance with HPAEC-PAD and gas chromatography.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Determination of the intracellular localization of SSI.Figure S2. Determination of GBSSI level.Figure S3. Expression and purification of recombinant SSI ex-pressed in E. coli.Figure S4. Determination of the transcription level of SSI in differentorgans of Arabidopsis.Figure S5. Evolution of the transcript level of SSI in Arabidopsisleaves during a 24 h cycle.

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