ldentification of the major starch synthase in the soluble ...the isoform accounts for -80% of the...

$: ldentification of the Major Starch Synthase in the Soluble ...the isoform accounts for -80% of the activity in the soluble fraction of the tuber and that it is also bound to starch$
The Plant Cell, Vol. 8, 1121-1 135, July 1996 O 1996 American Society of Plant Physiologists

ldentification of the Major Starch Synthase in the Soluble Fraction of Potato Tubers

Jacqueline Marshall,' Christopher Sidebottom,b Martine Debet,b Cathie Martin,' Alison M. Smith,'?' and Anne Edwards' a John lnnes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom

Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1 LQ, United Kingdom

The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA. The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described. lmmunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for -80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules. Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers. However, they caused a profound change in the morphology of starch granules, indicative of important under- lying changes in the structure of starch polymers within the granule.

INTRODUCTION

The storage organs of most species of plants contain multiple forms of starch synthases (Martin and Smith, 1995; Smith et al., 1995). The best understood of these isoforms belong to a widely distributed and highly conserved class of granule- bound starch synthases of -60 kD, collectively referred to as granule-bound starch synthase I (GBSSI; Martin and Smith, 1995). Studies of waxy, amf, and lam mutants of cereals, potatoes, and pea (Tsai, 1974; Hovenkamp-Hermelink et al., 1987; Denyer et al., 1995a) and potatoes in which activity of GBSSI was reduced through expression of antisense mRNA (Visser et al., 1991; Kuipers et al., 1994) have shown a positive corre- lation between GBSSl activity and the ratio of amylose to amylopectin in starch. When GBSSI is absent, starch contains only the highly branched polymer amylopectin. This suggests that GBSSl is responsible for synthesis of amylose, an almost unbranched polymer. The other isoforms of starch synthase, in conjunction with starch-branching enzyme, are presumably responsible for amylopectin synthesis. Amylopectin molecules have complex, genetically determined branching patterns, and it is not clear whether different isoforms of starch synthase make different contributions to this structure.

The first step in attempting to understand the functions of these starch synthases is to characterize all of the isoforms in one organ. A few isoforms of starch synthase other than GBSSl have been identified biochemically and molecularly in pea (Smith, 1990; Denyer and Smith, 1992; Dry et al., 1992) and rice (Baba et al., 1993), and biochemically in maize (Mu et al., 1994) and wheat (Denyer et al., 1995b). In most cases,

l To whom correspondence should be addressed.

the quantitative and qualitative contributions of characterized isoforms to starch synthesis have not been assessed, so a complete picture of the role and importance of all of the isoforms of starch synthase is not available for any storage organ.

Carbohydrate metabolism and starch synthesis have been studied extensively in the potato tuber (Geigenberger and Stitt, 1993; Hajirezaei et al., 1993; Geigenberger et al., 1994; Sonnewald et al., 1994), and this organ has great potential as a source of commercially important starches created through genetic manipulation (Shewmaker and Stalker, 1992; Visser and Jacobsen, 1993; Müller-Rober and Kossmann, 1994). One of the major gaps in understanding starch synthesis in this organ, and hence in the ability to manipulate its starch in use- ful ways, is the nature of its starch synthases. In potato, only two starch synthases (GBSSI and GBSSII) have been characterized in detail. Like other members of its class, GBSSI of potato is a granule-bound isoform responsible for the synthesis of amylose (Vos-Scheperkeuter et al., 1986; Hovenkamp- Hermelink et al., 1987; van der Leij et al., 1991). GBSSll is both granule bound and present in soluble form in the stroma of the amyloplast (Edwards et al., 1995); we refer to this isoform as SSll to reflect the fact that it is not exclusively granule bound. Its predicted amino acid sequence and molecular mass are similar to those of SSll (GBSSII) of pea embryos, an isoform of 77 kD that accounts for 60 to 70% of the starch synthase activity in the soluble fraction of the pea embryo (Denyer and Smith, 1992; Dry et al., 1992; Denyer et al., 1993). However, SSll accounts for only -10 to 15% of the total soluble starch synthase activity in potato tubers (Edwards et al., 1995). The isoform(s) responsible for most of the soluble activity of starch synthase in the potato tuber is thus unidentified.

1122 The Plant Cell

There have been severa1 attempts to characterize the starch synthases found in the soluble fraction of potato tubers and to purify the major soluble starch synthases (Frydman and Cardini, 1966; Hawker et al., 1972; Catz et al., 1989; Baba et al., 1990; Ponstein, 1990). However, varying numbers and molecular masses of isoforms of soluble starch synthases have been reported. The quantitative contribution of the putative forms has not been established, and when multiple forms are postulated, it is not clear whether they are modifications of a single gene product or the products of different genes. Forms that are products of different genes are more likely to have distinct properties and patterns of expression, and hence are more likely to play different roles in starch synthesis, than forms that are products of a single gene.

In this study, we describe the purification to homogeneity of an isoform of starch synthase (SSIII) from potato tubers and the preparation of a specific antiserum. lmmunoprecipitation experiments in conjunction with native gels established that SSlll is likely to account for most of the soluble activity in the tuber. To provide independent evidence about the quantitative importance of SSlll and to allow its role to be established, we used a cDNA clone obtained via the antibody to express antisense SSlll mRNA in transformed potatoes. The tubers of transformed lines displayed a severe and specific reduction in the amounts of SSlll protein and a reduction of up to 80% in total soluble starch synthase activity. There was little or no alteration in the starch content of the tubers or the amylose-to-amylopectin ratio of the starch, but the morphology of the granules was dramatically altered.

RESULTS

Starch Synthase Activity from the Soluble Fraction of Tubers Copurifies with Proteins of 110 and 120 kD

The starch synthase activity from the soluble fraction of developing tubers of cultivar Desiree and mature tubers of cultivar Estima eluted from both DEAE-Sepharose and Blue Sepharose columns as a single peak of activity. However, subsequent chromatography on a Mono Q column at pH 7.5 separated two major peaks of starch synthase activity, designated peak I and peak II according to their order of elution from the column (Figure 1). These two peaks of starch synthase activity were then purified separately by cyclohexaamylose and Mono Q chromatography. A typical purification from developing Desiree tubers is shown in Table 1. The specific activity of peak I was 5.1-pmol min-’ mg-l protein, a purification of 400-fold relative to the initial supernatant. The specific activity of peak II was 8.8 pmol min-’ mg-’ protein, a purification of 700-fold relative to the initial supernatant.

SDS-PAGE of the fractions from the final Mono Q column for ,peak I showed that the distribution of a protein of 120 kD matched the distribution of the starch synthase activity (Figures 2A and 26). SDS-PAGE of the fractions from the final Mono

800 I I 0.5 .- o 5 600 > m .-

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Fractions (1 ml)

Figure 1. Mono Q Chromatography of Partially Purified Starch Syn- thase Activity from lhe Soluble Fraction of Developing Desiree Tubers.

Partially purified starch synthase was subjected to Mono Q chromatography following DEAE-Sepharose and Blue Sepharose chromatography. The Mono Q column was eluted with a linear gradient of KCI (. . .). Samples of each 1-mL fraction were assayed for starch synthase activity ( -O- ) ; activity is expressed as nanomoles per minute per milliliter. Absorbance (280 nm) is indicated as a solid line, on a scale of O to 2 A. The peaks of activity designated I and II are indicated.

Q column for peak II showed that the distribution of the major protein of 110 kD exactly matched the starch synthase activity (Figures 2C and 2D).

Antiserum raised against GBSSl from pea embryos did not recognize any proteins from peak I or peak II. Antiserum raised against SSll from pea embryos very weakly recognized the 120- and 110-kD proteins from peaks I and II, respectively (data not shown).

An Antiserum Raised against Soluble Starch Synthase Recognizes Granule-Bound and Soluble Proteins of 140 kD

To obtain sufficient protein for preparation of an antiserum, peaks I and II from the initial Mono Q column were combined and purified together in large-scale preparations made from mature tubers of cultivar Estima. The final preparations (referred to as starch synthase III [SSlll]) usually contained proteins in addition to the 120- and 110-kD proteins, but these were minor components. 60th the 120- and 110-kD proteins were excised and eluted from gels of the final preparations and were injected into the same rat.

The antiserum raised against SSlll was used to probe blots of purified SSlll and extracts from mature Estima and developing Desiree tubers (Figure 3A). On immunoblots of the gels of the purified preparation of soluble starch synthase from mature Estima tubers, the antiserum recognized strongly the two proteins against which it was raised as well as a minor protein of 140 kD. It recognized a protein of 140 kD in all of the crude and partially purified preparations of both Estima and Desiree tubers against which it was tested. These included partially purified soluble starch synthase and granule-bound proteins

Soluble Starch Synthase of Potato Tubers 1123

from mature Estima tubers, and crude, soluble extracts andgranule-bound proteins from developing tubers of Desiree (Fig-ure 3A). In contrast, proteins of 120 and 110 kD were notdetected in most of these preparations. A protein of 120 kDwas weakly detectable in partially purified starch synthase frommature Estima tubers. These results suggest that the purified110- and 120-kD proteins may be derived from the 140-kD pro-tein during extraction and purification. Soluble extracts ofdeveloping Desiree tubers also contained a protein of lowermolecular mass (~105 kD) recognized by the antiserum (Fig-ure 3A, lane 6). The identity of this protein is not known. Onall of the immunoblots, the preimmune serum did not cross-react with any of the proteins.

The Antiserum Precipitates 75% of the Soluble StarchSynthase Activity of the Tuber

To determine whether the proteins recognized by the antise-rum raised against SSIII represent the major soluble starch

Table 1. Purification of Starch Synthase Activity from theSoluble Fractions of Developing Tubers of Cultivar Desiree3

205

Fraction

InitialSupernatant

0 to 40%(NH4)2S04DEAE-SepharoseBlue Sepharose

Peak lc

Mono Q (pH 7.5)CyclohexaamyloseMono Q (pH 8.0)

Peak ll°Mono Q (pH 7.5)CyclohexaamyloseMono Q (pH 8.0)

TotalActivity(limolGlucoseIncor-poratedmin~1)

28.9

17.1

7.514.59

0.950.530.15

2.452.270.26

ActivityRe-covered6

(%)

100

61.0

26.816.4

3.41.90.5

8.88.10.9

TotalProtein(mg)2211

1019

45.110.4

1.700.200.03

2.900.400.03

SpecificActivity(nmolGlucoseIncorporatedmin"1 mg~1

Protein)

0.013

0.017

0.1660.441

0.562.655.13

0.855.678.84

a Tubers (500 g) were homogenized, and the extract was filtered andcentrifuged. The supernatant was brought to 40% saturation with(NH4)2SO4, and the redissolved precipitate was subjected to chro-matography on columns of DEAE-Sepharose, Blue Sepharose, andMono Q (pH 7.5). After each step, the fractions containing the higheststarch synthase activity were pooled. The values are those of a typi-cal purification.b Activity recovered after each step is expressed as a percentage ofthat present in the initial supernatant.c The two peaks of starch synthase activity eluting from the Mono Qcolumn (peaks I and II) were purified separately by Cyclohexaamyloseand then Mono Q chromatography. The final Mono Q values are forthe single fraction with the highest starch synthase activity.

32 33 34 35 36 37 38 39 40 41Fraction (0.5 ml)

205.

116-»97-66.

25 26 27 28 29 30 31 32 33 34Fraction (0.5 ml)

Figure 2. Purification of Starch Synthase Activity from the SolubleFraction of Developing Desiree Tubers.

Peaks I and II from the initial Mono Q column (Figure 1) were chro-matographed separately on a column of cyclohexaamylose-Sepharosefollowed by a Mono Q column.(A) and (C) SDS-polyacrylamide gels of fractions in (B) and (D), respec-tively. Each lane contains 10 nL of the fraction. Positions of molecularmass standards loaded on the same gel are indicated at left in kilodal-tons. Arrowheads indicate the positions of the 120-kD (peak I) and110-kD (peak II) proteins that coeluted with starch synthase activity.(B) and (D) Peak I and peak II, respectively, showing starch synthaseactivity in the fractions indicated. Activity is expressed as nanomolesper minute per milliliter.

1124 The Plant Cell

1 2 3 4 5 6

7 8 9 10SSIII .H

SSII ••

5 10 15Volume of serum

SSIIISSII

GBSSI

Figure 3. Identification of SSIII Protein, Activity, and Transcript inDeveloping Tubers.

(A) Immunoblots of purified SSIII and tuber extracts. Lane 1 is anSDS-polyacrylamide gel of purified SSIII; lane 2, immunoblot of thegel in lane 1; lane 3, partially purified SSIII (see Methods) from maturetubers of cultivar Estima; lane 4, granule-bound proteins from Estimastarch; lane 5, granule-bound proteins from Desiree starch; and lane6, crude, soluble extract of freshly harvested, developing Desiree tubers.Immunoblots were developed with antiserum raised against SSIII ata dilution of 1/2500. Molecular masses in kilodaltons are indicated atleft. Lanes 7 to 10 are native glycogen-containing polyacrylamide gelsdeveloped for starch synthase activity, containing the supernatantsfrom incubations of crude, soluble extracts of freshly harvested, de-veloping Desiree tubers with antiserum raised against SSIII (lane 7),a mixture of antiserum raised against SSIII and antiserum raised againstSSII of pea embryos (lane 8), antiserum raised against SSII of peaembryos (lane 9), and a control incubation containing preimmune se-

synthase(s), the antiserum was used in immunoprecipitationexperiments with crude, soluble extracts from developingtubers of cultivar Desiree. Incubation of soluble extract withpreimmune serum from the rat did not affect soluble starchsynthase activity, but the antiserum raised against SSIII precipi-tated starch synthase activity (Figure 3B). The maximuminhibition of starch synthase activity was ~75°/o. Approximately30% of the remaining starch synthase activity can be ac-counted for by SSII (Table 2). When the soluble extract wasincubated with antiserum raised against SSII from pea em-bryos (which recognizes SSII in potato; Edwards et al., 1995),~9% of the starch synthase activity was inhibited. When theextract was incubated with both antisera, the starch synthaseactivity was reduced by ~80%.

Native PAGE of soluble extracts of developing tubers of cul-tivar Desiree revealed two major groups of bands with starchsynthase activity (Figure 3A). We have previously shownthrough antisense and immunoprecipitation experiments thatthe lower group of bands is attributable to SSII (Edwards etal., 1995). When the supernatant from the immunoprecipita-tion experiment with the rat antiserum raised against SSIII wassubjected to native PAGE, the upper group of bands was miss-ing but the lower group was unaffected (Figure 3A). Incubationwith the preimmune serum from rat had no effect on the bandsof starch synthase activity.

Isolation of a cDNA Clone for Soluble Starch Synthase

The results described above suggest that the isoform(s) rec-ognized by the antibody raised against SSIII accounts for 75%of the soluble starch synthase activity of the tuber. To provideindependent evidence about its importance and role, we iso-lated a cDNA clone by using the anti-SSIII antibody and thenused the clone as the basis for antisense constructs. The an-tibody was used to screen a Xgt11 oligo(dT)-primed libraryconstructed from poly(A)+ RNA from developing tubers of cul-tivar Desiree. The initial screen yielded a partial cDNA clone

rum from the rat subsequently immunized with SSIII (lane 10). Positionsof SSIII and SSII are indicated.(B) Immunoprecipitation of starch synthase activity from crude, solu-ble extracts of developing Desiree tubers with antiserum raised againstSSIII. Soluble extracts were incubated with increasing volumes of preim-mune serum (O) and antiserum (•), as described in Methods. Aftercentrifugation, the supernatant was assayed for starch synthase ac-tivity. Starch synthase activity is expressed as a percentage of activityof incubations containing 20 g L"1 BSA in PBS. Values are measure-ments from two separate experiments with independently preparedextracts. The line joins the mean values.(C) RNA gel blot of poly(A)+ RNA from a developing Desiree tuber (5ng per lane) probed with the 1.1-kb partial cDNA clone for SSIII (lane1), a cDNA clone for potato SSII (2.6-kb transcript, lane 2), and a cDNAclone for potato GBSSI (2.4-kb transcript, lane 3).


of 2.4 kb. Further screening of a random primed library with the 5' region of this clone yielded an overlapping clone of 2.3 kb. The full-length cDNA was 4.127 kb.

To check the identity of the cDNA, the amino acid sequence it predicted was compared with amino acid sequences of two peptides obtained by digestion with endoproteinase Lys-C of the 110-kD protein purified from tubers of cultivar Estima. The peptide sequences FIPIPYTSENVVEGK and HIPVFGG cor- responded precisely to predicted sequences from the clone. Attempts to obtain N-terminal amino acid sequence of the purified proteins for comparison with the sequence predicted from the cDNA clone were unsuccessful.

On RNA gel blots of poly(A)+ RNA from developing tubers, a partial cDNA clone recognized a single transcript of -4 kb. This size is considerably greater than those of the transcripts for GBSSl and SSll and is consistent with the transcript encoding a protein in the range of 110 to 140 kD (Figure 3C).

The deduced amino acid sequence of SSlll revealed a protein of 1230 amino acids and a predicted size of 139 kD (Figure 4). At the N terminus was a sequence of -60 amino acids rich in serine and basic residues and low in acidic residues, which is typical of a chloroplast transit peptide. Based on the con-

Table 2. lmmunoprecipitation of Starch Synthase Activity in a Crude, Soluble Extract from Developing Desiree Tubers and Tubers of Transformed Line 9 ~~~~ ~

lnhibition of Starch Synthase Activity (%)"

Serum in the lncubation Medium

Desiree Transformed Tuberb Tuber (Line 9)c

Preimmuned 0.3 f 0.9 0.1 Anti-potato SSllP 74 f 4 16

Anti-potato SSlll + anti-pea SSW 80 & 8 51

a After incubation, samples were centrifuged and the supernatant was assayed for starch synthase activity. Values are the percentage of inhibition relative to controls in which BSA at 20 g L-I in PBS was substituted for serum.

Values for Desiree tubers are the means 2 SE of measurements with extracts of four separate tubers. C Values for tubers of line 9 are the means of measurements with extracts of two tubers.

Preimmune: 1/10 dilution (final concentration in the incubation) of crude serum from the rat subsequently immunized with SSIII. eAnti-potato SSIII: 1/10 dilution of crude serum from the rat immunized with potato SSIII. Anti-pea SSII: 1/5 dilution of the immunoglobulin fraction of rabbit

serum containing the antibody raised against SSll from pea embryos. QAnti-potato SSlll + anti-pea SSII: a mixture of the two sera described above at dilutions of 1/10 and 1/5, respectively.

Anti-pea SSII' 9 f 4 39

sensus of Gavel and von Heinje (1990), the most likely cleavage site would be between amino acids 60 (Cys) and 61 (Ala), be- cause the serine-rich region ends before this point. Cleavage in this region would give a mature protein of -132 kD. The structure of SSlll is somewhat similar to that of SSll in that it contains a C-terminal region homologous with starch synthases and bacterial glycogen synthases and an N-terminal extension. The N-terminal extension shows little sequence similarity to the N-terminal extensions of SSll from pea or potato (in turn, they show little similarity to each other; Edwards et al., 1995) or to any other sequence in the data bases. The N-terminal domain resembles those of pea and potato SSll in that it shows considerable predicted flexibility (Chou-Fasman algorithm; see Dry et al., 1992); all these extensions may there- fore serve similar roles. At the C-terminal end of the N-terminal extension of SSlll are two proline residues; multiple proline residues have been noted previously at the C-terminal ends of N-terminal extensions of both starch synthases and starch- branching enzymes (Dry et al., 1992; Burton et al., 1995).

The roles of these N-terminal extensions are not known, but it seems likely that they are involved in determining properties such as interaction with starch polymers rather than contributing to basic catalytic properties. The C-terminal region of SSlll from amino acid 780 to the end shows greatest similarity to glycogen synthases from bacteria, although there is also significant similarity to other starch synthases from plants. The KTGG motif close to the N terminus of this region beginning with position 794 is conserved (KVGGL). This domain is thought to be involved in ADP/ADP-glucose binding (Furukawa et al., 1990). Interestingly, a second domain with a similar structure is also conserved in the C termini of all bac- teria1 glycogen synthases and starch synthases (including the motif beginning at position 1143, T/~GGLXDT1/~); this may represent a second domain involved in ADP/ADP-glucose binding. The whole region around this second domain is widely conserved among a-l,4-g1ucosyltransferases, indicating close involvement with the catalytic process.

Over the rest of the SSlll protein, there are severa1 other domains showing significant conservation between different starch synthases. However, SSlll also shows some notable gaps in its sequence when aligned with GBSSl and SSII, for example, between amino acids 828 to 829 (13 amino acids), 894 to 895 (10 amino acids), and 944 to 945 (35 amino acids). These regions may confer specific properties on GBSSl and SSll compared with SSIII.

Antisense Plants Have Strongly Reduced Soluble Starch Synthase Activity

Tuber discs of cultivar Desiree were transformed by using Agrobacterium containing a construct of the 1.1-kb partial cDNA clone encoding the C-terminal end of SSIII, in an antisense orientation under the control of a double cauliflower mosaic

i 126 The Plant Cell

SSIII M D V P F P L H R S L S C T S V S N A I T H L K I K P I L G F V S H G T T S L S V Q S S S W R K D G M V T G V S F S I C 60

SSIII A N F S G R R R R K V S T P R S Q G S S P K G F V P R K P S G M S T Q R K V Q K S N G D K E S K S T S T S K E S E I S N 1 2 0

SSIII Q K T V E A R V E T S D D D T K G V V R D H K F L E D E D E I N G S T K S I S M S P V R V S S Q F V E S E E T G G D D K 180

SSIII D A V K L N K S K R S E E S G F I I D S V I R E Q S G S Q G E T N A S S K G S H A V G T K L Y E I L Q V D V E P Q Q L K 2 4 0

SSIlI E N N A G N V E Y K G P V A S K L L E I T K A S D V E H T E S N E I D D L D T N S F F K S D L I E E D E P L A A G T V E 300

SSIII T G D S S L N L R L E M E A N L R R Q A I E R L A E E N L L Q G I R L F C F P E V V K P D E D V E I F L N R G L S T L K 360

SSIll N E S D V L I M G A F N E W R Y R S F T T R L T E T H L N G D W W S C K I H V P K E A Y R A D F V F F N G Q D V Y D N N 4 2 0

SSII . . , . . . . . . .

GBSSI . . . . . . . . . . . . . . . . . . M A V . 2;: ~ K , T L . 2 4 V N R K . 213 I K T A K E T K E R 659

GBSSI , _ . . . . . . . . . . , . . . . . . . . . . Q ~ G L . . R N H ~ L T H . . A V N K L D G L Q S T N T K V 56

SSllI T M R S F L L S Q K H V V Y T E P L D I Q A G S T Y Y N - A N U V L N G K ~ ! ~ ! ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ! f l ~ ;:i SsIl . . , . . . . . . . . . . . . . . . . . . . . I L R D R . . :flG K K I Q S Y M

GBSSI T P m A S R T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A T I V C G K G 80 SSII 0 S S A E A N . . . - . . . SSIII P Q m S P A B N G T H " R R T $ K " P G M D Y H I P V F ~ ~ ~ ~ : ~ ~ ~ ;;:

H C B K R a V

V L N L N S S N ~ F s%PYGE~;;P H V P C G G V C G D

F . . . . . . . . . . L L Q G F S P D I I H

;'IFSfSUg;; N D m D E F R G S D F I B G Y E K

F G A . . . _ , . . _ _ _ _ _ . _ , . . . , . . _ _ . _ . _ _ . . . . _ . . G P L E P H Y M D P n K L Y U . . . .a

. _ . . . . m G Y T G F H M G A F N V E C P A D m L K I V T T 541

. . . . _ . . Q P F D P L M S Q D W G P S D H D m R A Q Q C G L E PNiFSfI!a ~ ~ ~ & ~ ~ ~ t ~ ~ :?:6

L G L G A S G S E P G V E G E E I A P L A K 601 ;m a::::: : : : : : : : : : : : : : : E 0

GBSSI E N V A T P 607

Figure 4. Degree of Relatedness between SSlll and Other Starch Synthases.

Potato starch synthases were aligned using the PILEUP and PRETTYBOX programs (Devereux et ai., 1984). Solid black boxes indicate identical amino acids, heavily hatched boxes indicate similar amino acids, and lightly hatched boxes indicate related amino acids. The sequence of SSlll has EMBL accession number X95759.


C 5 19 25 2 9 18 26

Figure 5. Effects of Transformation with the Antisense Construct forSSIII on Levels of SSIII and GBSSI Transcripts in Tubers.

Shown are the results for tubers from individual, independently de-rived transgenic plants. Numbers assigned to these plants and thelines subsequently derived from them are indicated at bottom. Lanesdesignated C are control tubers from plants of cultivar Desiree grownin the same environment at the same time as the transgenic plants.(A) RNA gel blots probed with the 1.1-kb partial cDNA clone for SSIII.Each lane contains 50 ng of total RNA from a tuber taken from a grow-ing plant. Arrowheads indicate sense (upper) and antisense (lower)transcripts.(B) RNA gel blots shown in (A) reprobed with a cDNA clone for GBSSIof potato.

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virus 35S promoter, in pBIN19. Thirteen independently trans-formed plants and four independent control plants (transformedwith the vector alone) were transferred to a soil-based com-post and allowed to develop tubers. The presence of the SSIIIantisense construct was confirmed by DNA gel blotting (datanot shown). Six of the transgenic plants had levels of SSIIItranscript indistinguishable from those of the control plantson RNA gel blots. However, seven independent transformants(named 1, 2, 9, 18, 19, 25, and 26) had strongly reduced orundetectable levels of SSIII transcript (Figure 5A). The lossor reduction of detectable transcript was specific for SSIII, andthere was little variation in the level of transcript for GBSSIamong the plants studied (Figure 5B).

Tubers of the transformants with unaltered levels of SSIIItranscript had soluble starch synthase activities that were in-distinguishable from those of the control plants and from valuestypical of those obtained from developing Desiree tubers ingeneral (Edwards et al., 1995). Tubers of the seven transfor-mants with reduced or undetectable levels of SSIII transcripthad significantly reduced activities, and three plants displayedactivities that were 30% or less of the average value of thecontrol plants. Figure 6A shows values from an initial screenof single tubers harvested from actively growing plants (valuesare means of measurements made with two replicate samplesof each tuber). Table 3 shows that the reductions in solublestarch synthase activity seen in this initial screen were repro-ducible from one tuber to another. They were also reproduciblethrough tuber development.

Figure 6. Effects of Transformation with the Antisense Construct forSSIII on Soluble Starch Synthase Activity and Starch Content of Tubers.

Shown are the results for tubers from individual, independently de-rived transgenic plants. Numbers assigned to these plants and thelines subsequently derived from them are indicated at bottom. C, con-trol tubers from individual plants of cultivar Desiree grown in the sameenvironment at the same time as the transgenic plants.(A) Soluble starch synthase activity. Values are in nanomoles per minuteper gram fresh weight of tissue and are the means of measurementsmade with two replicate samples of tissue taken from a core throughthe center of a tuber (between 12 and 50 g fresh weight) from a grow-ing plant.(B) Native glycogen-containing polyacrylamide gels developed forstarch synthase activity, containing the soluble extracts used for mea-surements of starch synthase activity in (A). Note the absence of theupper SSIII band (see Figure 2A, lanes 7 to 10) in the right-hand lanes.Differences between lanes in the mobilities of the lower bands aredue to differences between individual gels and not to differences be-tween plants. Arrowheads indicate bands attributable to SSII (lower)and SSIII (upper).(C) Starch contents of actively growing tubers measured on the in-soluble fractions of the extracts used for measurements of starchsynthase activity in (A). Values are in milligrams per gram fresh weightof tissue and are the means of measurements made with two sepa-rate extracts.(D) Starch contents of mature tubers measured on cores of ~2 g oftissue taken from the centers of tubers at the time of harvest. Valuesare in milligrams per gram fresh weight of tissue and are means ofmeasurements made on two to three tubers for each plant. Plants forwhich no data are presented were not sampled.Bars in (A), (C), and (D) that represent control plants are hatched forease of reference.

1128 The Plant Cell

Table 3. Effects of Reduced Activity of SSIIIGranule-Bound Starch Synthase Activity andof Starch

Granule-BoundSoluble Activity" Activity"(nmol min~' g~' (nmol min"1 g~'

Plant3 Fresh Weight) Fresh Weight)

1 ND° NO2 ND ND9 18.3 ± 3.9 (4) 118

18 23.6 ± 6.7 (3) 9725 29.5 ± 3.6(4) 11326 33.3 ± 8.3 (3) 80Control 98.4 ± 4.9(9) 106 ± 12Desiree ND ND

on Soluble andAmylose Content

AmyloseContent"1

(% Total Starch)

27.829.5, 29.828.629.327.330.126.4, 28.927.8, 29.2

a Plant numbers refer to individual transgenic plants with reducedSSIII activity. Tubers are from a single plant, except for the controlline, in which three different plants (each an independent, control trans-formant) were used.b Soluble activity was measured by using duplicate samples fromtubers of 12 to 70 g fresh weight harvested at intervals during plantdevelopment. Values are the means ±SE of measurements madewith the number of tubers given within parentheses.c Granule-bound activities are the means of measurements made byusing duplicate samples from a single tuber (12 to 70 g fresh weight)harvested at maturity.d Amylose content was measured by using starch extracted from twoor three tubers per mature plant. Values are the means of measure-ments made with two separate samples taken from the bulk starchpreparations: two values are given when independent starch prepa-rations were used. Wild-type Desiree plants used for these measure-ments were grown in the same greenhouse at the same time as thetransgenic plants.e ND, not determined.

Reductions in Starch Synthase Activity AreSpecifically Due to Loss of SSIII

To discover whether the reductions in activity were specificallyattributable to loss of SSIII, two sorts of experiments were un-dertaken. First, isoforms were visualized on native gels of crude,soluble extracts of transformed tubers. The group of bandsattributable to SSIII was present in extracts from control plantsand from all six of the transformants with soluble starch syn-thase activities comparable with control activities. It was absentfrom extracts of all seven transformants with reduced starchsynthase activities. Other groups of bands on the gels, includ-ing those attributable to SSII, were present in all extracts (Figure6B). Second, crude, soluble extracts from a plant with stronglyreduced activity were incubated with the antiserum raisedagainst SSIII. The antiserum inhibited activity by 16%, com-pared with 75% inhibition in extracts of untransformed tubersof cultivar Desiree (Table 2).

Loss of starch synthase activity from the soluble fraction intransgenic tubers was accompanied by dramatic reductionsin the amount of the 140-kD protein recognized by the antise-

rum in soluble and granule-bound fractions of the tuber. Theprotein was not detected, or detected only very weakly rela-tive to controls, on immunoblots of these fractions from tubersof the six transgenic lines with the largest reductions in starchsynthase activity (representative results in Figures 7A and 7D).In contrast, the soluble protein of 105 kD also recognized bythe antiserum was present in equal amounts in all lines exam-ined (Figure 7A).

B

180 •-SSIII -

84 »•

180SSIII-

84 *•

Figure 7. Effects of Transformation with the Antisense Construct forSSIII on SSIII, GBSSI, and SSII Proteins.

Numbers below the gels indicate the transgenic line represented ineach lane. C, control plants.(A) Immunoblot of a gel of crude, soluble extracts of developing Desireetubers, using an antiserum (1/1000 dilution) raised against SSIII. Posi-tions of SSIII and of molecular mass standards in kilodaltons areindicated at left. The band at 105 kD in all three lanes is the unknownprotein that is recognized by the antiserum raised against SSIII.(B) Immunoblot of a gel of crude, soluble extracts of developing Desireetubers, using an antiserum (1/1000 dilution) raised against SSII of peaembryos.(C) SDS-polyacrylamide gel of granule-bound proteins. The positionof GBSSI is indicated at left.(D) Immunoblot of a gel of granule-bound proteins, using an antise-rum (1/1000 dilution) raised against SSIII. Positions of SSIII and ofmolecular mass standards in kilodaltons are indicated at left.(E) Immunoblot of a gel of granule-bound proteins, developed withan antiserum (1/1000 dilution) raised against SSII of pea embryos.


Reduction in SSIII Activity Alters Granule Shape butHas Little Effect on Starch and Amylose Content

Tubers of the seven transformants with reduced activities ofsoluble starch synthase had starch granules with strikingly al-tered morphology. Two types of granule were present: simplegranules with deep, often T-shaped cracks centered on thehilum, and granules that appeared to be large clusters of tiny,spherical granules. A range of different sizes of both types ofgranule was present in tubers at various developmental stages(Figure 8).

In spite of the alteration in granule morphology, tubers oftransformants with reduced activity of SSIII were indistinguish-able from control tubers with respect to starch content. Thiswas true of both developing tubers and tubers of mature plantson which the haulm was senescent (Figures 6C and 6D). Thestarch of these plants also displayed no significant alterationin amylose content (Table 3).

Reduction in SSIII Activity Does Not Affect OtherIsoforms of Starch Synthase

We considered the possibility that the reduction in SSIII in trans-formed tubers has secondary effects on other isoforms of starchsynthase. Any alterations in other isoforms could seriously af-fect deductions about the importance and role of SSIII. Effectsof the reduction in SSIII on GBSSI were assessed by measur-ing granule-bound starch synthase activity in crude extractsof tubers and examining gels of granule-bound proteins. Therewas no difference in granule-bound activity between controlplants and those in which soluble starch synthase activity wasreduced (Table 3). More than 95% of the starch synthase ac-tivity of intact starch granules of wild-type potatoes isattributable to GBSSI (Edwards et al., 1995). Reductions inSSIII also had no obvious effect on the amount of GBSSI pro-tein bound to starch granules (Figure 7C).

Effects of reductions in SSIII on SSII were assessed in threeways. First, amounts of SSII protein in the soluble and granule-bound fractions of the tuber were visualized by immunoblot-ting. There were no obvious differences between control plantsand those in which SSIII was reduced (Figures 7B and 7E).Second, as described above, SSII was visualized on nativegels of crude, soluble extracts stained for starch synthase ac-tivity. Again, there were no marked or consistent differencesbetween control plants and those in which SSIII was reduced(Figure 6B). Neither of the above-mentioned methods providesquantitative information about the contribution of SSII to starchsynthase activity. To provide this, immunoprecipitation experi-ments were used to assess the proportion of the residualactivity attributable to SSII in tubers in which SSIII was reduced.The antiserum raised against SSII of pea embryos, whichrecognizes SSII of potatoes (Edwards et al., 1995), inhibited~40% of the activity in tubers in which soluble starch synthaseactivity was reduced by ^80% (line 9) compared with 9% incontrol and wild-type tubers (Table 2). Using these figures andstarch synthase activities from Table 3, the activity attributable

Figure 8. Effect of Reduced SSIII on the Morphology of StarchGranules.

Shown is photomicroscopy of starch from developing tubers, viewedwith a phase-contrast microscope.(Top) Cultivar Desiree.(Bottom) Transgenic plant 1. Bar = 20 urn.

to SSII is 7.3 nmol min~1 g~1 fresh weight in line 9 and 8.8nmol min~1 g~1 fresh weight in control tubers. This indicatesthat the reduction in SSIII has little effect on the soluble activ-ity of SSII.

DISCUSSION

Our purification procedure for potato tubers identified two solu-ble proteins, of 110 and 120 kD, as starch synthases. Thespecific activities of these proteins (5.1 and 8.8 nmol min'1mg~1 protein, respectively) are seven- to 300-fold higher thanthose of the partially purified starch synthases from potatotubers reported by Hawker et al. (1972) (0.64 nmol min~1 mg~1

protein), Baba et al. (1990) (0.03 nmol min"1 mg~1 protein), andPonstein (1990) (1.35 and 0.91 nmol min'1 mg~1 protein) andare comparable to those of starch synthases from the solublefractions of other storage organs. Purification to nearhomogeneity resulted in specific activities of 16 nmol min"1

mg~1 protein for SSII from pea embryos (Denyer and Smith,1992), 14 nmol min"1 mg~1 protein for an SSII-like isoform

1130 The Plant Cell

from wheat endosperm (Denyer et al., 1995b), and 9 pmol min-’ mg-I protein for a major isoform from maize endosperm (Mu et al., 1994).

Although our purification procedure yielded active starch synthases of 110 and 120 kD, it seems likely that these are breakdown products of a 140-kD isoform. First, the antibody raised against these proteins recognized a 140-kD protein in extracts of potato; this protein is also a minor component of the purified SSlll preparations. The 110- and 120-kD proteins either were not detected or were detected to a much lesser extent than the 140-kD protein in crude extracts. Second, expression of antisense mRNA from the partia1 cDNA clone isolated via the antibody resulted in loss of the 140-kD protein from all of the plants displaying reduced starch synthase activity. The identity of amino acid sequences from the 110-kD protein and predicted sequences from the cDNA clone showed that the clone encodes at least one of the two purified proteins. The existence of small amounts of the 120-kD protein in extracts of mature tubers suggests that some breakdown of the 140-kD protein may occur in vivo, perhaps during storage of tubers. The smaller starch synthase proteins purified from potato tubers by other workers (see Introduction) may be further breakdown products of the 140-kD isoform, generated during tuber storage or purification or both.

SSlll is distinct from GBSSI and SSII, the two isoforms previously characterized from potato tubers. It is only very weakly recognized by an antibody that strongly recognizes SSII, and its predicted amino acid sequence differs substantially from those of the other two isoforms. The amino acid sequence is -30% identical and 50% similar (assessed according to Schwartz and Dayhoff [1979]) to those of both GBSSI and SSII. It is also distinctly different from all starch synthases for which sequence information is available and from bacterial glycogen synthases. It is actually less similar to other plant isoforms than to the bacterial enzymes and may thus be a more an- cient form of starch synthase than the other isoforms characterized so far (Figure 9). The fact that isoforms similar to SSlll have not previously been described is likely to reflect the paucity of information about starch synthases generally rather than the rarity of isoforms of this type. Most of the starch synthases for which information is available belong to the GBSSI class: currently, there are only three sequences for isoforms that are at least partly soluble. Probably much of the soluble activity in many organs is contributed by isoforms distinctly different from these three (e.g., wheat endosperm; Denyer et al., 1995b), and isoforms similar to SSlll may well prove to be widespread.

Two independent lines of evidence show that SSlll contributes most of the soluble starch synthase activity of the potato tuber. lmmunoinhibition experiments with the antibody raised against SSlll indicate that it accounts for -75% of the soluble activity. Consistent with this result, the maximum reduction of soluble starch synthase activity achieved via antisense transformation experiments was 70 to 80%. lmmunoinhibition experiments and native activity gels on extracts of tubers of the antisense transformants confirmed that the reduction in

Agro GS Ecoli GS Bsub GS Maize GBSSI Rice GBSSI

Wheat GBSSI Barley GBSSI Cassava GBSSI Potato GBSSI Pea GBSSI Potato SSll

Pea SSll

Rice SSS Potato SSlll

Figure 9. Relationships between Starch Synthases and Bacterial Glycogen Synthases.

The dendrogram was generated using the PILEUP program (Devereux et al., 1984). The identities of the proteins are as follows: Agro GS, Agrobacterium glycogen synthase (Uttaro et al., 1990); Ecoli GS, €scherichia coli glycogen synthase (Kumar et al., 1986); Bsub GS, Bacillus subtilis glycogen synthase (Kiel et al., 1994); maize GBSSl (Klosgen et al., 1986); rice GBSSI (Okagaki, 1992); wheat GBSSI (Clark et al., 1991); barley GBSSI (Rohde et al., 1988); cassava GBSSI (Salehuzzaman et al., 1993); potato GBSSI (Visser et al., 1989); pea GBSSI (Dry et al., 1992); potato SSll (Edwards et al., 1995); pea SSll (Dry et al., 1992); rice SSS, rice soluble starch synthase (Baba et al., 1993); and potato SSlll (this study).

activity was specifically due to loss of SSIII. lmmunoblotting experiments showed that SSlll is also present on starch granules, but it is likely to contribute only a tiny percentage of the granule-bound activity. At least 95% of the activity of intact granules is contributed by GBSSI, and SSII- which is a much more abundant granule-bound protein than SSIII-probably contributes most of the remainder.

Although SSll contributes some of the soluble starch synthase activity not attributable to SSIII, other unknown isoforms may also be present. lmmunoinhibition experiments with SSlll and SSll antisera on wild-type tubers and transgenic tubers with reduced levek of SSlll consistently suggest that -15% of the soluble activity is not attributable to either of these isoforms.

Reductions in soluble starch synthase activity of up to 70 to 80% had no measurable effect on the starch content of the tuber either during growth or at maturity. The lowest activities of soluble starch synthase in transgenic tubers are comparable with estimated rates of starch accumulation in tubers (32 to 47 nmol of glucose units min-’ g-’ fresh weight; Morrell and ap Rees, 1986). Soluble starch synthase activity is proposed to be of major importance in controlling flux through the path- way of starch synthesis in developing wheat endosperm (Jenner et al., 1993; Keeling et al., 1993); our results indicate that this is highly unlikely to be the case in potato tubers. How- ever, it is unlikely that our measurements of starch content are


adequate to detect subtle differences between control and transgenic tubers in the rate of starch synthesis in particular parts of the tuber or at particular stages of development.

The dramatic effect of reductions in SSlll on the morphology of the starch granule indicates that important changes in the structure of starch have occurred. Although both fissured and apparently compound granules are present, the latter could be derived by tangential swelling of wedge-shaped parts of the granule created by deep fissuring. The appearance of deep fissures during granule growth is also observed in embryos of the r mutant of pea. This mutant has very low activity of starch-branching enzyme and hence an increased amylose content and amylopectin with greater average branch lengths (Colonna and Mercier, 1984; Smith, 1988; Bhattacharyya et al., 1990). Reduced swelling of these granules, leading to fissuring, has been attributed to their high amylose content (Colonna and Mercier, 1985). Although reduced swelling may cause the fissuring of granules from our transgenic tubers, this cannot be attributed to a change in amylose content of the starch. Detailed structural analysis of starch from the transgenic tubers should provide new insights into factors that determine the gelatinization properties of starch granules.

Changes in starch structure are likely to be the direct result of the reduction in SSlll rather than the result of secondary changes in other isoforms of starch synthase. The amount and activity of GBSSI and SSII, and the contribution to activity of isoforms that remain to be characterized, are not obviously affected by reductions in the amount of SSIII. A reduction in SSlll will result in changes in the ratio of the soluble fraction of starch synthase activity to activities of both starch-branching enzyme and granule-bound starch synthases, and either or both of these changes would be expected to alter starch structure. Further workon possible effectsof the reduction in SSlll on the activities of other enzymes of starch synthesis during tuber development and on the structure of starch is in progress.

METHODS

Plant Material

Tubers of potato (Solanum tuberosum) cultivars Desiree and Estima were used. Results obtained with the two cultivars were identical. Desiree tubers were grown in pots of soil-based compost (25 cm in diameter) in a greenhouse at a minimum temperature of 12"C, with supplementary lighting in winter. They were freshly harvested from actively growing plants immediately before experiments. Estima tubers were bought locally.

Purification of Soluble Starch Synthase

All procedures were performed at O to 4°C.

Small-Scale Procedures

Approximately 500 g of Desiree tubers was homogenized in an elec- tric blender with 25 g of polyvinylpolypyrrolidone and 500 mL of medium A (100 mM Tris-HCI, pH 7.5, 10 mM EDTA, 5 mM DTT, 1 g L-l sodium metabisulfite, 0.5 mg L-' leupeptin, 0.7 mg L-' pepstatin A, 50 mL L-' glycerol). The homogenate was filtered through two layers of muslin and centrifuged at l0,OOOg for 10 min, and the supernatant was brought to 40% saturation with solid (NH4)2S04. The precipitate was redissolved in a minimal volume of medium A and dialyzed for3 hr against 2 L of medium A.

The extract was applied, at a flow rate of 4 mL min-I, to a column (5 cm i.d.; 10 cm long) of DEAE-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden), which was equilibrated with medium A. The column was washed with 500 mL of medium A, followed by a 250-mL gradient of O to 1 M KCI in the same medium. Fractions(10 mL) with the highest starch synthase activity were pooled and dialyzed overnight against 5 L of medium B (50 mM Tris-HCI, pH 7.5, 1 mM EDTA, 1 mM DTT, 0.5 mg L-I leupeptin, 0.7 mg L-I pepstatin A, 50 mL L-I glycerol).

The extract was applied, at a flow iate of 1 mL min-l, to a column (1.6 cm i.d.; 16 cm long) of Blue Sepharose (Sigma), which was equilibrated with medium B. The column was washed with 100 mL of medium B, followed by a 100-mL gradient of O to 1 M KCI in the same medium. Fractions (5 mL) with the highest starch synthase activity were pooled and dialyzed for 3 hr against 5 L of medium 6.

The extract was applied, at a flow rate of 0.5 mL min-l, to a I-mL Mono Q column (Pharmacia), which was equilibrated with medium B. The column was washed with 25 mL of medium B, followed by a 25-mL gradient of O to 0.5 M KCI in the same medium. Fractions (0.5 mL) from each of two peaks of activity were pooled and purified separately as follows.

After mixing with an equal volume of 1 M sodium citrate in medium 6 (pH adjusted to 7.5), the Mono Q eluate was applied, at a flow rate of 0.5 mL min-l, to a column (1.0 cm i.d.; 4 cm long) of cyclohexaamylose-Sepharose (prepared according to Vretblad [1974]), which was equilibrated with 0.5 M sodium citrate in medium B. The column was washed with 20 mL of medium B containing 0.5 M sodium citrate, and the protein was eluted with 30 mL of medium 6 containing no citrate. Fractions (1 mL) with the highest starch synthase activity were pooled and dialyzed for a minimum of 3 hr against 5 L of medium C (50 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT, 0.5 mg L-l leupeptin, 0.7 mg L-l pepstatin A, 50 mL L-I glycerol).

The extract was applied, at a flow rate of 0.5 mL min-l, to a I-mL Mono Q column equilibrated with medium C. The column was washed with 25 mL of medium C followed by a 25-mL gradient of O to 0.5 M KCI in the same medium. Fractions (0.5 mL) were assayed for starch synthase activity.

Large-Scale Procedures

The procedures were as described above, with the following modifications. Five kilograms of Estima tubers was homogenized in 5 L of medium A with 250 g of polyvinylpolypyrrolidone, filtered, and centrifuged at 10,OOOg for 10 min. Polyethylene glycol 6000 (500 g L-1 in medium A) was added to the supernatant to a concentration of 100 g L-'. The precipitate was redissolved in a minimal volume of medium A.

The extract was mixed for 1 hr with a 900-mL slurry of DEAE- Sepharose equilibrated with medium A. The DEAE-Sepharose was washed with 2 L of medium A, incubated for 1 hr in 500 mL of medium A containing 400 mM KCI, and then washed with an additional 500 mL of this solution. The washes containing KCI were combined and

1132 The Plant Cell

brought to 50% saturation with solid (NH&SO.,. The precipitate was redissolved in a minimal volume of medium B and dialyzed for a minimum of 5 hr against 5 L of medium B.

The extract was applied, at a flow rate of 2 mL min-l, to a Blue Sepharose column (5 cm i.d.; 15 cm long) equilibrated with medium B. The column was washed with 300 mL of medium B, followed by a 600-mL gradient of O to 1 M KCI in the same medium, at a flow rate of 5 mL min-l. Fractions (15 mL) with the highest starch synthase activity were pooled and dialyzed overnight against 5 L of medium B. The dialyzed eluate was applied to a 1-mL Mono Q column, as described above, except that all of the fractions containing starch synthase activity were pooled. The eluate was applied to a cyclohexaamylose-Sepharose column (1.0 cm i.d.; 20 cm long) as described above. The column was washed with 50 mL of medium B containing 0.5 M sodium citrate and eluted with 80 mL of medium B without citrate. Frac- tions with starch synthase activity were pooled and dialyzed against medium C. The dialyzed extract was applied to a 1-mL MonoQ column equilibrated with medium C, as described above. Fractions containing starch synthase activity were stored at -2OOC.

SDS-PAGE and lmmunoblotting

Protein samples were dialyzed against distilled water and then mixed 1:l with double-strength sample buffer (Laemmli, 1970) and boiled for 2 min immediately before application to gels. Granule-bound proteins were prepared, and gels were run according to Edwards et al. (1995). Gels were stained with Coomassie Brilliant Blue R. lmmunoblots were prepared and developed according to Bhattacharyya et al. (1990). The nitrocellulose filters were incubated either with crude rat serum followed by alkaline phosphatase-conjugated goat anti-rat antiserum (Sigma) or with the immunoglobulin fraction of rabbit serum raised against starch synthase II (SSII) from pea embryos (Smith, 1990), followed by alkaline phosphatase-conjugated goat anti-rabbit antiserum (Sigma).

Determination of Amino Acid Sequence

Protein from SDS-polyacrylamide gels was prepared and sequenced as described by Denyer et al. (1993).

Preparation of Antiserum Native PAGE

Protein from five large-scale purifications was subjected to SDS-PAGE, as described below. Starch synthase proteins were electroeluted, dialyzed against water, and freeze-dried. Protein (50 pg) was redissolved in 250 pL of PBS, mixed with 250 pL of Freund’s complete adjuvant, and injected intramuscularly into a rat. Subsequent injections were of 75 pg of protein dissolved in 250 pL of PBS mixed with 250 pL of Freund’s incomplete adjuvant and were repeated at 14-day intervals. Serum was collected from 14 days after the third injection.

Assay of Soluble Starch Synthase Activity

Soluble starch synthase activity was measured using the resin method described in Jenner et al. (1994).

Preparation of Crude, Soluble Extracts of Potato Tuber

Representative samples (0.5 to 2.0 g of fresh weight) were taken from cores through the tuber at a point approximately half way between the dista1 and proximal ends of the tuber. The outer 1 to 2 mm of the tuber was removed from the ends of the core before sampling. Sam- ples were homogenized in 4volumes of 50 mM Tris-HCI, pH 7.5, 1 mM EDTA, 1 mM DTT, 1 g L-’ sodium metabisulfite, 0.5 mg L-l leupeptin, 0.7 mg L-I pepstatin A, and 50 mL L-l glycerol at O°C and then centrifuged at 10,000gfor 10 min. The supernatant is referred to as soluble extract .

Partia1 Purification of Soluble Starch Synthase Activity

Crude, soluble extracts of mature Estima tubers (5 to 10 g of fresh weight of tissue) were dialyzed for 3 hr against 5 L of medium B at 4OC and applied to a 1-mL Mono Q column equilibrated with medium 6, as described above. The fraction with the highest starch synthase activity (referred to as partially purified soluble starch synthase) was stored at -2OOC.

Sample preparation, electrophoresis, and gel development were as described by Edwards et al. (1995).

lmmunoprecipitation

Soluble extracts (100 pL) were incubated with O to 20 pL rat serum or 20 pL of the immunoglobulin fraction of rabbit serum to SSll from pea embryos (Smith 1990) for 1.5 hr at room temperature on a rotating table. Twenty microliters of rabbit antiserum raised against rat IgG at 2.5 g L-’ specific antibody (Sigma) was then added to samples containing rat serum, and incubation continued for another 0.5 hr. To all samples, 50 pL of protein A-Sepharose at 60 g L-l in 50 mM Tris-HCI, pH 7.5, was added and then incubated for 0.5 hr, followed by centrifu- gation at 10,OOOg for 10 min. The supernatants were assayed for starch synthase activity. Controls contained BSA at 20 g L-l in PBS in place of serum.

lsolation and Analysis of Starch Granules

Purified starch was prepared from potato tubers as described by Edwards et al. (1995). Amylose content was measured by a colorimetric, iodine-based assay as described by Morrison and Laignelet (1983).

Measurement of Protein

Protein was assayed using a protein assay dye reagent (Bio-Rad) with a standard curve of BSA.

lsolation of cDNA Clones

hgtll libraries were kindly provided by C. Grierson (John lnnes Centre). The antiserum raised against the purified starch synthase proteins from


tubers of cultivar Estima was used in the immunoscreening of an am- plified Lgtl l library containing cDNA inserts with EcoRl linkers, prepared from cDNA of developing tubers. Approximately 1.5 x 106 plaque-forming units were probed with the antiserum ata dilution of 111000. The second antibody was an anti-rat immunoglobulin linked to horseradish peroxidase (Amersham International, Amersham, UK). Two positive clones were isolated. These were both 1.1 kb in length and contained poly(A) tracts at their 3'ends. One of these was cloned into the EcoRl site of pBluescript SK+ to give plasmid pRAT2. A 5' EcoRI-EcoRV fragment from this clone was used as a probe on the kgtl l library. Filters were washed in 0.1 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate), 0.5 g L-I SDS at 65OC. Seven clones of 1.3, 1.53, 1.75, 1.88, 2.15,2.21, and 2.4 kbwere isolated. The longest clone was subcloned as an EcoRl fragment into pBluescript SK+ to give plasmid pRAT20. A 600-bp 5' fragment from pRAT20 was used to probe a random primed hgtl l library prepared from cDNA of developing tubers. Three positive clones were isolated. The longest was 2.3 kb and was subcloned as an EcoRl fragment into pBluescript SK+ to give pRAT24.

DNA sequences were determined according to Sanger et al. (1977) by using Sequenase (United States Biochemical). Sequence data were analyzed using the Genetics Computer Group (Madison, WI) computer program (Devereux et al., 1984).

Construction of Antisense Binary Vector

The 1.1-kb Pst-EcoRV fragment from pRAT2 encoding the 5' end of SSlll was subcloned in an antisense orientation between the cauliflower mosaic virus double 35s promoter and cauliflower mosaic virus terminator (Pstl-Smal) in pJlT60 (Guerineau and Mullineaux, 1993), producing pRAT3. The Xhol-partia1 Sstl fragment from pRAT3, encom- passing the promoter, antisense cDNA, and terminator, was ligated between the Sall-Sstl sites of the plant transformation vector pBIN19 (Bevan, 1984), resulting in plasmid pRAT4.

Transformation of Potato

Binary plasmid pRAT4 was introduced into Agrobacterium tumefaciens by the freeze-thaw method of An et al. (1988). Preparation of Agrobac- terium inoculum carrying the antisense consiruct, inoculation of tuber discs of potato cultivar Desiree, regeneration of shoots, and rooting of shoots were as described by Edwards et al. (1995).

Preparation of DNA, RNA and mRNA, Gel Blot Analysis, and Radiolabeling of Probes

All methods were as described by Edwards et al. (1995). Filters were stripped before reprobing by washing in 0.1 x SSC, 5 g L-' aqueous SDS for 30 min at 70°C. The probe for potato granule-bound starch synthase I (GBSSI) was a full-length cDNA clone isolated from a tuber cDNA library with a pea GBSSI probe (Dry et al., 1992).

ACKNOWLEDGMENTS

This work was funded by the Biotechnology and Biological Sciences Research Council of the United Kingdom and by Unilever plc through

a LlNK grant. We thank Dr. Kay Denyer (John lnnes Centre) for her advice throughout the courseof this work and Drs. Mike Gidley, Steve Jobling, and Dick Safford (Unilever Research) for their helpful com- ments. We are particularly grateful to Drs. Jens Kossmann and Gernot Abel (Max-Planck lnstitut für Molekulare Pflanzenphysiologie, Golm, Germany) for making unpublished data available to us.

Received February 26, 1996; accepted April 30, 1996.

ir

REFERENCES

An, G., Ebert, P.R., Mitra, A., and Ha, S.B. (1988). Binary vectors. In Plant Molecular Biology Manual A3, S.B. Gelvin and R.A. Schilperoort, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 1-19.

Baba, T., Noro, M., Hiroto, M., and Arai, Y. (1990). Properties of primer- dependent starch synthesis catalysed by starch synthase from potato tubers. Phytochemistry 29, 719-723.

Baba, T., Nishihara, M., Mizuno, K., Kawasaki, T., Shimada, H., Kobayashi, E., Ohnishi, S., Tanaka, K., and Arai, Y. (1993). Iden- tification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds. Plant Physiol.

Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711-8721.

Bhattacharyya, M.K., Smith, A.M., Ellis, T.H.N., Hedley, C., and Martin, C. (1990). The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell 60, 115-122.

Burton, R.A., Bewley, J.D.,Smith, A.M., Bhattacharyya, M.K., Tatge, H., Ring, S., Bull, V., Hamilton, W.P.D., and Martin, C. (1995). Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development. Plant

Catz, D.S., Moreno, S., and Tandecarz, J.S. (1989). Comparison of soluble starch synthetase patterns in tubers and tuber-derived calli of Solanum tuberosum L. An. Asoc. Quim. Argentina 77, 47-51.

Clark, J.R., Robertson, M., and Ainsworth, C.C. (1991). Nucleotide sequence of a wheat (Trticum aestivum) cDNA clone encoding the waxy protein. Plant MOI. Biol. 16, 1099-1101.

Colonna, P., and Mercier, C. (1984). Macromolecular structure of wrinkled- and smooth-pea starch components. Carbohydr. Res. 126,

Colonna, F!, and Mercier C. (1985). Gelatinization and melting of maize and pea starches with normal and high-amylose genotypes. Phytochemistry 24, 1667-1674.

Denyer, K., and Smith, A.M. (1992). The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos. Planta 186, 609-617.

Denyer, K., Sidebottom, C., Hylton, C.M., and Smith, A.M. (1993). Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos. Plant

Denyer, K., Barber, L.M., Burton, R., Hedley, C.L., Hylton, C.M., Johnson, S., Jones, D.A., Manhall, J., Smith, A.M., Tatge, H.,

103, 565-573.

J. 7,'3-35.

233-247.

J. 4, 191-198.

1134 The Plant Cell

Tomlinson, K., and Wang, T.L. (1995a). The isolation and characterisation of novel low-amylose mutants of Pisum sativum L. Plant Cell Environ. 18, 1019-1026.

Denyer, K., Hylton, C.M., Jenner, C.F., and Smith, A.M. (1995b). ldentification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm. Planta 186, 256-265.

Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehen- sive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395.

Dry, I., Smith, A., Edwards, A., Bhattacharyya, M., Dunn, P., and Martin, C. (1992). Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato. Plant J. 2,

Edwards, A., Marshall, J., Sidebottom, C., Visser, R.G.F., Smith, A.M., and Martin; C. (1995). Biochemical and molecular characterization of a novel starch synthase from potato tubers. Plant J. 8,

Frydman, R.B., and Cardini, C.E. (1966). Studies on the biosynthesis of starch. I. lsolation and properties of the soluble adenosine diphosphate g1ucose:starch glucosyltransferase of Solanum tuberosum. Arch. Biochem. Biophys. 116, 9-18.

Furukawa, K., Tagaya, M., Inoye, M., Preiss, J., and Fukui, T. (1990). ldentification of lysine 15 at the active site in Escherichia coliglyco- gen synthase. J. Biol. Chem. 265, 2086-2090.

Gavel, Y., and von Heinje, G. (1990). A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett. 261, 455-458.

Geigenberger, P., and Stitt, M. (1993). Sucrose synthase catalyses a readily reversible reaction in vivo in developing potato tubers and other plant tissues. Planta 189, 329-339.

Geigenberger, P., Merlo, L., Reimholz, R., and Stitt, M. (1994). When growing potato tubers are detached from their mother plant there is a rapid inhibition of starch synthesis, involving inhibition of ADP-glucose pyrophosphorylase. Planta 193, 486-493.

Guerineau, F., and Mullineaux, P. (1993). Plant transformation and expression vectors. In Plant Molecular Biology Labfax, R.R.D. Croy, ed (Oxford, UK: BlOS Scientific Publishers), pp. 121-148.

Hajirezaei, M., Sonnewald, U., Viola, R., Carlisle, S., Dennis, D.T., and Stitt, M. (1993). Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotrans- ferase show no visible phenotype and only minor changes in tuber metabolism. Planta 192, 16-30.

Hawker, J.S., Ozbun, J.L., and Preiss, J. (1972). Unprimed starch synthesis by soluble ADPglucose-starch glucosyltransferase from potato tubers. Phytochemistry 11, 1278-1293.

Hovenkamp-Hermelink, J.H.M., Jacobsen, E., Ponstein, A.S., Visser, R.G.F., Vos-Scheperkeuter, G.H., Bijmolt, E.W., de Vries, J.N., Witholt, B., and Feenstra, W.J. (1987). lsolation of an amylose- free starch mutant of the potato (Solanum tuberosum L.). Theor. Appl. Genet. 75, 217-221.

Jenner, C.F., Siwek, K., and Hawker, J.S. (1993). The synthesis of [14C]starch from ['4C]sucrose in isolated wheat grains is dependent upon the activity of soluble starch synthase. Aust. J. Plant Physiol.

Jenner, C.F., Denyer, K., and Hawker, J.S. (1994). Caution on the use of the generally accepted methanol precipitation technique for the assay of soluble starch synthase in crude extracts of plant tissues. Aust. J. Plant Physiol. 21, 17-22.

193-202.

283-294.

20, 329-335.

Keeling, P.L., Bacon, P.J., and Holt, D.C. (1993). Elevated temperature reduces starch deposition in wheat endosperm by reducing the activity of soluble starch synthase. Planta 191, 342-348.

Kiel, J.A., Boels, J.M., Beldman, G., and Venema, G. (1994). Glyco- gen in Bacillus subtilis: Molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degra- dation. MOI. Microbiol. 11, 203-218.

Klosgen, R.B., Gierl, A., Schwarz-Sommer, Z., and Saedler, H. (1986). Molecular analysis of the waxy locus of maize. MOI. Gen. Genet. 203, 237-244.

Kuipers, A.G.J., Jacobsen, E., and Visser, R.G.F. (1994). Formation and deposition of amylose in the potato tuber starch granule are affected by the reduction of granule-bound starch synthase gene expression. Plant Cell 6, 43-52.

Kumar, A., Larsen, C.E., and Preiss, J. (1986). Biosynthesis of bac- teria1 glycogen: Primary structure of Escherichia coli ADP-glucose: a-l,4-glucan 4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene. J. Biol. Chem. 261, 16256-16259.

Laemmli, U.K. (1970). Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature 227, 680-6135,

Martin, C., and Smith, A.M. (1995). Starch biosynthesis. Plant Cell

Morrell, S., and ap Rees, T. (1986). Sugar metabolism in developing tubers of Solanum tuberosum. Phytochemistry 25, 1579-1585.

Morrison, W.R., and Laignelet, 8. (1983). An improved colorimetric procedure for determining apparent and total amylose in cereal and other starches. J. Cereal Sci. 1, 9-20.

Mu, C., Harn, C., Ko, Y.T., Singletary, G.W., Keeling, P.L., and Wasserman, B.P. (1994). Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv 673) endosperm. Plant

MÜller-Rober, B., and Kossmann, J. (1994). Approaches to influence starch quantity and starch quality in transgenic plants. Plant Cell Environ. 17, 601-613.

Okagaki, R.J. (1992). Nucleotide sequence of a long cDNA from the rice waxy gene. Plant MOI. Biol. 19, 513-516.

Ponstein, A. (1990). Starch Synthesis in Potato Tubers. PhD Disser- tation (Groningen, The Netherlands: State University of Groningen).

Rohde, W., Becker, D., and Salamini, F. (1988). Structural analysis of the waxy locus from Hordeum vulgare. Nucleic Acids Res. 16, 7185-7186.

Salehuuaman, S.N.I.M., Jacobsen, E., and Visser, R.G.F. (1993). lsolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato. Plant MOI. Biol. 23, 947-962.

Sanger, R., Nicklen, S., and Coulson, A.R. (1977). DNA sequenc- ing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,

Schwartz, R.M., and Dayhoff, M.O. (1979). Matrices for detecting dis- tant relationships. In Atlas of Protein Sequence and Structure, M.O. Dayhoff, ed (Washington, DC: National Biomedical Research Foun- dation), pp. 353-358.

Shewmaker, C.K., and Stalker, D.M. (1992). Modifying starch biosynthesis with transgenes in potatoes. Plant Physiol. 100, 1083-1086.

Smith, A.M. (1988). Major differences in isoforms of starch-branching enzyme in embryos of round- and wrinkled-seeded peas (Pisum sativum L.). Planta 175, 270-279.

7, 971-985.

J. 6, 151-159.

5463-5467.


Smith, A.M. (1990). Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major granule-bound starch synthase. Planta

Smlth, A.M., Denyer, K., and Martin, C.R. (1995). What controls the amount and structure of starch in storage organs? Plant Physiol. 107,673-677.

Sonnewald, U., Lerchl, J., Zrenner, R., and Frommer, W. (1994). Manipulation of sink-source relations in transgenic plants. Plant Cell Environ. 17, 649-658.

Tsai, C X (1974). The function of the waxy locus in starch synthesis in maize endosperm. Biochem. Genet. 11, 83-96.

Uttaro, A.D., Cangelosi, G.A., Geremia, R.A., Nester, E.W., and Ugalde, R.A. (1990). Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens. J. Bacteriol. 172,1640-1646.

van der Leij, F.R., Visser, R.G.F., Ponsteln, AS., Jacobsen, E., and Feenstra, W.J. (1991). Sequence of the structural gene for granule- bound starch synthase of potato (Solanum tuberosum L.) and evi- dente for a single point deletion in the amfallele. MOI. Gen. Genet.

182, 599-604.

228, 240-248.

Visser, R.G.F., and Jacobsen, E. (1993). Towards modifying plants for altered starch content and composition. Trends Biotech. 11,63-68.

Visser, R.G.F., Hergersberg, M., van der Leij, F.R., Jacobsen, E., Witholt, E., and Feenstra, W.J. (1989). Molecular cloning and par- tia1 characterisation of the gene for granule-bound starch synthase from a wild-type and an amylose-free potato (Solanum tuberosum L.). Plant Sci. 64, 185-192.

Visser, R.G.F., SOmhOKt, I., Kuipen, G.J., Ruys, N.J., Feenstra, W.J., and Jacobsen, E. (1991). lnhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. MOI. Gen. Genet. 225, 289-296.

Vos-Scheperkeuter, G.H., de Boer, W., Visser, R.G.F., Feenstra, W.J., and Witholt, B. (1986). ldentification of granule-bound starch synthase in potato tubers. Plant Physiol. 82, 411-416.

Vretblad, P. (1974). lmmobilization of ligands for biospecific affinity chromatography via their hydroxyl groups. The cyclohexaamylose-p- amylase system. FEBS Lett. 47, 86-89.

DOI 10.1105/tpc.8.7.1121 1996;8;1121-1135Plant Cell

J Marshall, C Sidebottom, M Debet, C Martin, A M Smith and A EdwardsIdentification of the major starch synthase in the soluble fraction of potato tubers.

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