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

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1 The Plant Cell, Vol. 8, , 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 underlying 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 granulebound 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 correlation 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 useful 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.

2 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 o II.- > m 2 - O I &* (u c c I x rn * I I I I.:. I *.e O 8 m - I \ -0.1 g r;; I.. Y..- Li ** O0 5 lb 15 +? 2 Fractions (1 ml) Figure 1. Mono Q Chromatography of Partially Purified Starch Synthase 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 -

3 Soluble Starch Synthase of Potato Tubers 1123 from mature Estima tubers, and crude, soluble extracts and granule-bound proteins from developing tubers of Desiree (Figure 3A). In contrast, proteins of 120 and 110 kd were not detected in most of these preparations. A protein of 120 kd was weakly detectable in partially purified starch synthase from mature Estima tubers. These results suggest that the purified 110- and 120-kD proteins may be derived from the 140-kD protein during extraction and purification. Soluble extracts of developing Desiree tubers also contained a protein of lower molecular mass (~105 kd) recognized by the antiserum (Figure 3A, lane 6). The identity of this protein is not known. On all of the immunoblots, the preimmune serum did not crossreact with any of the proteins. 205 The Antiserum Precipitates 75% of the Soluble Starch Synthase Activity of the Tuber To determine whether the proteins recognized by the antiserum raised against SSIII represent the major soluble starch Table 1. Purification of Starch Synthase Activity from the Soluble Fractions of Developing Tubers of Cultivar Desiree 3 Fraction Initial Supernatant 0 to 40% (NH 4 ) 2 S0 4 DEAE-Sepharose Blue Sepharose Peak l c Mono Q (ph 7.5) Cyclohexaamylose Mono Q (ph 8.0) Peak ll Mono Q (ph 7.5) Cyclohexaamylose Mono Q (ph 8.0) Total Activity (limol Glucose Incorporated min~ 1 ) Activity Recovered 6 (%) Total Protein (mg) Specific Activity (nmol Glucose Incorporated min" 1 mg~ 1 Protein) a Tubers (500 g) were homogenized, and the extract was filtered and centrifuged. The supernatant was brought to 40% saturation with (NH 4 ) 2 SO 4, and the redissolved precipitate was subjected to chromatography on columns of DEAE-Sepharose, Blue Sepharose, and Mono Q (ph 7.5). After each step, the fractions containing the highest starch synthase activity were pooled. The values are those of a typical purification. b Activity recovered after each step is expressed as a percentage of that present in the initial supernatant. c The two peaks of starch synthase activity eluting from the Mono Q column (peaks I and II) were purified separately by Cyclohexaamylose and then Mono Q chromatography. The final Mono Q values are for the single fraction with the highest starch synthase activity » Fraction (0.5 ml) Fraction (0.5 ml) Figure 2. Purification of Starch Synthase Activity from the Soluble Fraction of Developing Desiree Tubers. Peaks I and II from the initial Mono Q column (Figure 1) were chromatographed separately on a column of cyclohexaamylose-sepharose followed by a Mono Q column. (A) and (C) SDS-polyacrylamide gels of fractions in (B) and (D), respectively. Each lane contains 10 nl of the fraction. Positions of molecular mass standards loaded on the same gel are indicated at left in kilodaltons. Arrowheads indicate the positions of the 120-kD (peak I) and 110-kD (peak II) proteins that coeluted with starch synthase activity. (B) and (D) Peak I and peak II, respectively, showing starch synthase activity in the fractions indicated. Activity is expressed as nanomoles per minute per milliliter.

4 1124 The Plant Cell SSIII.H SSII (A) Immunoblots of purified SSIII and tuber extracts. Lane 1 is an SDS-polyacrylamide gel of purified SSIII; lane 2, immunoblot of the gel in lane 1; lane 3, partially purified SSIII (see Methods) from mature tubers of cultivar Estima; lane 4, granule-bound proteins from Estima starch; lane 5, granule-bound proteins from Desiree starch; and lane 6, crude, soluble extract of freshly harvested, developing Desiree tubers. Immunoblots were developed with antiserum raised against SSIII at a dilution of 1/2500. Molecular masses in kilodaltons are indicated at left. Lanes 7 to 10 are native glycogen-containing polyacrylamide gels developed for starch synthase activity, containing the supernatants from incubations of crude, soluble extracts of freshly harvested, developing Desiree tubers with antiserum raised against SSIII (lane 7), a mixture of antiserum raised against SSIII and antiserum raised against SSII of pea embryos (lane 8), antiserum raised against SSII of pea embryos (lane 9), and a control incubation containing preimmune sesynthase(s), the antiserum was used in immunoprecipitation experiments with crude, soluble extracts from developing tubers of cultivar Desiree. Incubation of soluble extract with preimmune serum from the rat did not affect soluble starch synthase activity, but the antiserum raised against SSIII precipitated starch synthase activity (Figure 3B). The maximum inhibition of starch synthase activity was ~75 /o. Approximately 30% of the remaining starch synthase activity can be accounted for by SSII (Table 2). When the soluble extract was incubated with antiserum raised against SSII from pea embryos (which recognizes SSII in potato; Edwards et al., 1995), ~9% of the starch synthase activity was inhibited. When the extract was incubated with both antisera, the starch synthase activity was reduced by ~80%. Native PAGE of soluble extracts of developing tubers of cultivar Desiree revealed two major groups of bands with starch synthase activity (Figure 3A). We have previously shown through antisense and immunoprecipitation experiments that the lower group of bands is attributable to SSII (Edwards et al., 1995). When the supernatant from the immunoprecipitation experiment with the rat antiserum raised against SSIII was subjected to native PAGE, the upper group of bands was missing but the lower group was unaffected (Figure 3A). Incubation with the preimmune serum from rat had no effect on the bands of starch synthase activity. Isolation of a cdna Clone for Soluble Starch Synthase SSIII SSII GBSSI Volume of serum The results described above suggest that the isoform(s) recognized by the antibody raised against SSIII accounts for 75% of the soluble starch synthase activity of the tuber. To provide independent evidence about its importance and role, we isolated a cdna clone by using the anti-ssiii antibody and then used the clone as the basis for antisense constructs. The antibody was used to screen a Xgt11 oligo(dt)-primed library constructed from poly(a) + RNA from developing tubers of cultivar Desiree. The initial screen yielded a partial cdna clone Figure 3. Identification of SSIII Protein, Activity, and Transcript in Developing Tubers. rum from the rat subsequently immunized with SSIII (lane 10). Positions of SSIII and SSII are indicated. (B) Immunoprecipitation of starch synthase activity from crude, soluble extracts of developing Desiree tubers with antiserum raised against SSIII. Soluble extracts were incubated with increasing volumes of preimmune serum (O) and antiserum ( ), as described in Methods. After centrifugation, the supernatant was assayed for starch synthase activity. Starch synthase activity is expressed as a percentage of activity of incubations containing 20 g L" 1 BSA in PBS. Values are measurements from two separate experiments with independently prepared extracts. The line joins the mean values. (C) RNA gel blot of poly(a) + RNA from a developing Desiree tuber (5 ng per lane) probed with the 1.1-kb partial cdna clone for SSIII (lane 1), a cdna clone for potato SSII (2.6-kb transcript, lane 2), and a cdna clone for potato GBSSI (2.4-kb transcript, lane 3).

5 ~~~~ ~ Soluble Starch Synthase of Potato Tubers 1125 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 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 corresponded 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 Serum in the lncubation Medium lnhibition of Starch Synthase Activity (%)" Desiree Transformed Tuberb Tuber (Line 9)c Preimmuned 0.3 f Anti-potato SSllP 74 f 4 16 Anti-pea SSII' 9f4 39 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. sensus of Gavel and von Heinje (1990), the most likely cleavage site would be between amino acids 60 (Cys) and 61 (Ala), because 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 therefore 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 starchbranching 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 bacteria1 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

6 i 126 The Plant Cell SSIII MDVPFPLHRS LSCTSVSNAI THLKIKPILG FVSHGTTSLS VQSSSWRKDG MVTGVSFSIC 60 SSIII ANFSGRRRRK VSTPRSQGSS PKGFVPRKPS GMSTQRKVQK SNGDKESKST STSKESEISN 120 SSIII QKTVEARVET SDDDTKGVVR DHKFLEDEDE INGSTKSISM SPVRVSSQFV ESEETGGDDK 180 SSIII DAVKLNKSKR SEESGFIIDS VIREQSGSQG ETNASSKGSH AVGTKLYEIL QVDVEPQQLK 240 SSIlI ENNAGNVEYK GPVASKLLEI TKASDVEHTE SNEIDDLDTN SFFKSDLIEE DEPLAAGTVE 300 SSIII TGDSSLNLRL EMEANLRRQA IERLAEENLL QGIRLFCFPE VVKPDEDVEI FLNRGLSTLK 360 SSIll NESDVLIMGA FNEWRYRSFT TRLTETHLNG DWWSCKIHVP KEAYRADFVF FNGQDVYDNN 420 SSII.., GBSSI M A V. 2;: ~ K, T VN RK. L IKT A K ETKER 659 GBSSI,_....., Q ~ G L. RNH~LTH.. AVNKLD GLQS TNTKV 56 SsIl.., ILRDR.. :flg K K I Q S Y M ~ ~ ~ ~ ~ ~ ~! ~! ~ ;:i! ~ SSllI TMRSFLLSQK HVVYTEPLDI QAG S T YY N-ANUVLNGK GBSSI T P m A S R T ATIVCGKG 80 SSII 0 S S A E A N SSIII PQmSPABNG TH"RRT$K"P GMDYHIP V F ~ ~ ~ ~ : ~ ~ ;;: H C BK RaV VLNLNSSN~F s%pyge~;;p HVPCGGVC G D F..... LL Q GFSPDIIH NDmDEFRGS DFIBGYEK ;'IFSfSUg; FGA..._,..._,...,..._..._.. GPLE PHYMDPn KLYU....a GBSSI E N V A T P 607 Figure 4. Degree of Relatedness between SSlll and Other Starch Synthases.._... mgytgfhmg AFNVEC P A DmL K I V T T _..QPFDPLMSQ DW GPS DHD mraqqcgle PNiFSfI!a ~ ~ :?:6 ~ & ~ LG LGASGSEPGV EGEEIAPLAK 601 ;m a::::: : : : : : : : : : : : : : : E 0 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.

7 Soluble Starch Synthase of Potato Tubers 1127 A I 60 < 40 B 20 o C Figure 5. Effects of Transformation with the Antisense Construct for SSIII on Levels of SSIII and GBSSI Transcripts in Tubers. Shown are the results for tubers from individual, independently derived transgenic plants. Numbers assigned to these plants and the lines subsequently derived from them are indicated at bottom. Lanes designated C are control tubers from plants of cultivar Desiree grown in 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 growing plant. Arrowheads indicate sense (upper) and antisense (lower) transcripts. (B) RNA gel blots shown in (A) reprobed with a cdna clone for GBSSI of potato in oo CM en CM r- virus 35S promoter, in pbin19. Thirteen independently transformed plants and four independent control plants (transformed with the vector alone) were transferred to a soil-based compost and allowed to develop tubers. The presence of the SSIII antisense construct was confirmed by DNA gel blotting (data not shown). Six of the transgenic plants had levels of SSIII transcript indistinguishable from those of the control plants on RNA gel blots. However, seven independent transformants (named 1, 2, 9, 18, 19, 25, and 26) had strongly reduced or undetectable levels of SSIII transcript (Figure 5A). The loss or reduction of detectable transcript was specific for SSIII, and there was little variation in the level of transcript for GBSSI among the plants studied (Figure 5B). Tubers of the transformants with unaltered levels of SSIII transcript had soluble starch synthase activities that were indistinguishable from those of the control plants and from values typical of those obtained from developing Desiree tubers in general (Edwards et al., 1995). Tubers of the seven transformants with reduced or undetectable levels of SSIII transcript had significantly reduced activities, and three plants displayed activities that were 30% or less of the average value of the control plants. Figure 6A shows values from an initial screen of single tubers harvested from actively growing plants (values are means of measurements made with two replicate samples of each tuber). Table 3 shows that the reductions in soluble starch synthase activity seen in this initial screen were reproducible from one tuber to another. They were also reproducible through tuber development. Figure 6. Effects of Transformation with the Antisense Construct for SSIII on Soluble Starch Synthase Activity and Starch Content of Tubers. Shown are the results for tubers from individual, independently derived transgenic plants. Numbers assigned to these plants and the lines subsequently derived from them are indicated at bottom. C, control tubers from individual plants of cultivar Desiree grown in the same environment at the same time as the transgenic plants. (A) Soluble starch synthase activity. Values are in nanomoles per minute per gram fresh weight of tissue and are the means of measurements made with two replicate samples of tissue taken from a core through the center of a tuber (between 12 and 50 g fresh weight) from a growing plant. (B) Native glycogen-containing polyacrylamide gels developed for starch synthase activity, containing the soluble extracts used for measurements of starch synthase activity in (A). Note the absence of the upper SSIII band (see Figure 2A, lanes 7 to 10) in the right-hand lanes. Differences between lanes in the mobilities of the lower bands are due to differences between individual gels and not to differences between plants. Arrowheads indicate bands attributable to SSII (lower) and SSIII (upper). (C) Starch contents of actively growing tubers measured on the insoluble fractions of the extracts used for measurements of starch synthase activity in (A). Values are in milligrams per gram fresh weight of tissue and are the means of measurements made with two separate extracts. (D) Starch contents of mature tubers measured on cores of ~2 g of tissue taken from the centers of tubers at the time of harvest. Values are in milligrams per gram fresh weight of tissue and are means of measurements made on two to three tubers for each plant. Plants for which no data are presented were not sampled. Bars in (A), (C), and (D) that represent control plants are hatched for ease of reference.

8 1128 The Plant Cell Table 3. Effects of Reduced Activity of SSIII Granule-Bound Starch Synthase Activity and of Starch Granule-Bound Soluble Activity" Activity" (nmol min~' g~' (nmol min" 1 g~' Plant 3 Fresh Weight) Fresh Weight) 1 ND NO 2 ND ND ± 3.9 (4) ± 6.7 (3) ± 3.6(4) ± 8.3 (3) 80 Control 98.4 ± 4.9(9) 106 ± 12 Desiree ND ND on Soluble and Amylose Content Amylose Content" 1 (% Total Starch) , , , 29.2 a Plant numbers refer to individual transgenic plants with reduced SSIII activity. Tubers are from a single plant, except for the control line, in which three different plants (each an independent, control transformant) were used. b Soluble activity was measured by using duplicate samples from tubers of 12 to 70 g fresh weight harvested at intervals during plant development. Values are the means ±SE of measurements made with the number of tubers given within parentheses. c Granule-bound activities are the means of measurements made by using 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 two or three tubers per mature plant. Values are the means of measurements made with two separate samples taken from the bulk starch preparations: two values are given when independent starch preparations were used. Wild-type Desiree plants used for these measurements were grown in the same greenhouse at the same time as the transgenic plants. e ND, not determined. To discover whether the reductions in activity were specifically attributable to loss of SSIII, two sorts of experiments were undertaken. First, isoforms were visualized on native gels of crude, soluble extracts of transformed tubers. The group of bands attributable to SSIII was present in extracts from control plants and from all six of the transformants with soluble starch synthase activities comparable with control activities. It was absent from extracts of all seven transformants with reduced starch synthase activities. Other groups of bands on the gels, including those attributable to SSII, were present in all extracts (Figure 6B). Second, crude, soluble extracts from a plant with strongly reduced activity were incubated with the antiserum raised against SSIII. The antiserum inhibited activity by 16%, compared with 75% inhibition in extracts of untransformed tubers of cultivar Desiree (Table 2). Loss of starch synthase activity from the soluble fraction in transgenic tubers was accompanied by dramatic reductions in the amount of the 140-kD protein recognized by the antiserum in soluble and granule-bound fractions of the tuber. The protein was not detected, or detected only very weakly relative to controls, on immunoblots of these fractions from tubers of the six transgenic lines with the largest reductions in starch synthase activity (representative results in Figures 7A and 7D). In contrast, the soluble protein of 105 kd also recognized by the antiserum was present in equal amounts in all lines examined (Figure 7A) SSIII - 84» 180 SSIII- 84 * B Reductions in Starch Synthase Activity Are Specifically Due to Loss of SSIII Figure 7. Effects of Transformation with the Antisense Construct for SSIII on SSIII, GBSSI, and SSII Proteins. Numbers below the gels indicate the transgenic line represented in each lane. C, control plants. (A) Immunoblot of a gel of crude, soluble extracts of developing Desiree tubers, using an antiserum (1/1000 dilution) raised against SSIII. Positions of SSIII and of molecular mass standards in kilodaltons are indicated at left. The band at 105 kd in all three lanes is the unknown protein that is recognized by the antiserum raised against SSIII. (B) Immunoblot of a gel of crude, soluble extracts of developing Desiree tubers, using an antiserum (1/1000 dilution) raised against SSII of pea embryos. (C) SDS-polyacrylamide gel of granule-bound proteins. The position of GBSSI is indicated at left. (D) Immunoblot of a gel of granule-bound proteins, using an antiserum (1/1000 dilution) raised against SSIII. Positions of SSIII and of molecular mass standards in kilodaltons are indicated at left. (E) Immunoblot of a gel of granule-bound proteins, developed with an antiserum (1/1000 dilution) raised against SSII of pea embryos.

9 Soluble Starch Synthase of Potato Tubers 1129 Reduction in SSIII Activity Alters Granule Shape but Has Little Effect on Starch and Amylose Content Tubers of the seven transformants with reduced activities of soluble starch synthase had starch granules with strikingly altered morphology. Two types of granule were present: simple granules with deep, often T-shaped cracks centered on the hilum, and granules that appeared to be large clusters of tiny, spherical granules. A range of different sizes of both types of granule was present in tubers at various developmental stages (Figure 8). In spite of the alteration in granule morphology, tubers of transformants with reduced activity of SSIII were indistinguishable from control tubers with respect to starch content. This was true of both developing tubers and tubers of mature plants on which the haulm was senescent (Figures 6C and 6D). The starch of these plants also displayed no significant alteration in amylose content (Table 3). Reduction in SSIII Activity Does Not Affect Other Isoforms of Starch Synthase We considered the possibility that the reduction in SSIII in transformed tubers has secondary effects on other isoforms of starch synthase. Any alterations in other isoforms could seriously affect deductions about the importance and role of SSIII. Effects of the reduction in SSIII on GBSSI were assessed by measuring granule-bound starch synthase activity in crude extracts of tubers and examining gels of granule-bound proteins. There was no difference in granule-bound activity between control plants and those in which soluble starch synthase activity was reduced (Table 3). More than 95% of the starch synthase activity of intact starch granules of wild-type potatoes is attributable to GBSSI (Edwards et al., 1995). Reductions in SSIII also had no obvious effect on the amount of GBSSI protein bound to starch granules (Figure 7C). Effects of reductions in SSIII on SSII were assessed in three ways. First, amounts of SSII protein in the soluble and granulebound fractions of the tuber were visualized by immunoblotting. There were no obvious differences between control plants and those in which SSIII was reduced (Figures 7B and 7E). Second, as described above, SSII was visualized on native gels of crude, soluble extracts stained for starch synthase activity. Again, there were no marked or consistent differences between control plants and those in which SSIII was reduced (Figure 6B). Neither of the above-mentioned methods provides quantitative information about the contribution of SSII to starch synthase activity. To provide this, immunoprecipitation experiments were used to assess the proportion of the residual activity attributable to SSII in tubers in which SSIII was reduced. The antiserum raised against SSII of pea embryos, which recognizes SSII of potatoes (Edwards et al., 1995), inhibited ~40% of the activity in tubers in which soluble starch synthase activity was reduced by ^80% (line 9) compared with 9% in control and wild-type tubers (Table 2). Using these figures and starch synthase activities from Table 3, the activity attributable Figure 8. Effect of Reduced SSIII on the Morphology of Starch Granules. Shown is photomicroscopy of starch from developing tubers, viewed with 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 8.8 nmol min~ 1 g~ 1 fresh weight in control tubers. This indicates that the reduction in SSIII has little effect on the soluble activity of SSII. DISCUSSION Our purification procedure for potato tubers identified two soluble proteins, of 110 and 120 kd, as starch synthases. The specific activities of these proteins (5.1 and 8.8 nmol min' 1 mg~ 1 protein, respectively) are seven- to 300-fold higher than those of the partially purified starch synthases from potato tubers 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), and Ponstein (1990) (1.35 and 0.91 nmol min' 1 mg~ 1 protein) and are comparable to those of starch synthases from the soluble fractions of other storage organs. Purification to near homogeneity 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

10 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 ancient 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 pathway 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. However, it is unlikely that our measurements of starch content are

11 Soluble Starch Synthase of Potato Tubers 1131 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 electric 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

12 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. Fractions 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. Preparation of Antiserum 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. Samples 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. 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). Native PAGE 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 centrifugation 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

13 Soluble Starch Synthase of Potato Tubers 1133 tubers of cultivar Estima was used in the immunoscreening of an amplified Lgtll 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 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 kgtll library. Filters were washed in 0.1 x SSC (1 x SSC is 0.15 M NaCI, 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 hgtll 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, encompassing 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 Agrobacterium 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 comments. 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, REFERENCES ir 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 Baba, T., Noro, M., Hiroto, M., and Arai, Y. (1990). Properties of primerdependent starch synthesis catalysed by starch synthase from potato tubers. 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