# Toward an understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas soluble starch synthase II controls the synthesis of intermediate size glucans of amylopectin.

Thierry Fontaine, C d'Hulst, M Maddelein, F Routier, T Pépin, A Decq, J Wieruszeski, B Delrue, N van den Koornhuyse, J Bossu

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Thierry Fontaine, C d'Hulst, M Maddelein, F Routier, T Pépin, et al.. Toward an understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas soluble starch synthase II controls the synthesis of intermediate size glucans of amylopectin.. Journal of Biological Chemistry, 1993, 268 (22), pp.16223-30. 10.1016/S0021-9258(19)85409-1. hal-04748947

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# Toward an Understanding of the Biogenesis of the Starch Granule

EVIDENCE THAT *CHLAMYDOMONAS* SOLUBLE STARCH SYNTHASE II CONTROLS THE SYNTHESIS OF INTERMEDIATE SIZE GLUCANS OF AMYLOPECTIN*

(Received for publication, December 7, 1992, and in revised form, March 10, 1993)

Thierry Fontaine‡, Christophe D'Hulst‡, Marie-Lise Maddelein, Françoise Routier, Thérèse Marianne Pépin, André Decq, Jean-Michel Wieruszeski, Brigitte Delrue, Nathalie Van den Koornhuyse, Jean-Pierre Bossu, Bernard Fournet, and Steven Ball§

From the Laboratoire de Chimie Biologique, Unité Mixte de Recherche du Centre National de la Recherche Scientifique 111, Université des Sciences et Technologies de Lille Flandres-Artois, 59655 Villeneuve, d'Ascq Cedex, France

Low starch mutants of *Chlamydomonas reinhardtii* were isolated after x-ray mutagenesis of wild-type strain 137C. The mutants accumulated 20-40% of the normal amount and displayed a 2-fold decrease of the total glycogen-primed soluble starch synthase activity. Three different mutant alleles of the *st-3* gene were isolated that were characterized by similar defects and displayed a net increase in amylose content. Amylose-primed synthesis of glucan in native gels revealed a complete wipe out of one of the soluble starch syntheses. Zymograms and kinetic analyses performed both in the mutant and in partially purified wild type extracts reveal at least two distinct activities that are partly analogous to higher plant soluble starch syntheses I and II (SSI and II). The *st-3* mutants were defective for SSII. Methylation and debranching of the purified amylopectin fraction clearly show a decrease in the number of intermediate size glucans (dp8 to 50) and an absolute and relative increase of very short glucans (dp2 to 7). These results suggest that a soluble starch synthase may be necessary for the synthesis or maintenance of intermediate size glucans that are the main component of the branched clusters of amylopectin.

Starch, the storage carbohydrate of most higher and lower plants, occurs as a large complex insoluble granule located both in leaf chloroplasts and in the amyloplasts of storage organs. It contains at least two types of polysaccharides: amylopectin can be defined as a very high molecular weight (~10⁷ to 10⁸) α-1,4-linked glucan with a significant number (5%) of α-1,6-linkages giving a moderately branched structure (for a review, see Manners, 1989). Amylose is usually defined as a smaller (M~r~ ~10⁵ to 10⁶) α-1,4-linked glucan with a significantly lower number of branches. Amylopectin, usually the most abundant of the two fractions is considered the backbone of the granule whereas amylose fits in, in some still unknown fashion. Our present limited understanding of the biogenesis of the starch granule's complicated structure stems

essentially from genetic and biochemical investigations performed in higher plants of which maize has undoubtedly been the most thoroughly studied (for reviews, see Preiss, 1991; Preiss and Levi, 1976; Shannon and Garwood 1984). From a biochemical point of view, 4-ADP-glucose:1,4-α-D-glucosyl-transferases (starch synthases) (Ozbun et al., 1971; Macdonald and Preiss, 1983, 1985) and at least two or three branching enzymes (1,4-α-D-glucan: 1,4-α-D-glucan 6-glucosyl-transferases) (Boyer and Preiss, 1978; Singh and Preiss, 1985) have been described in the maize endosperm. Two of the reported starch synthases have been shown to be associated with the starch granule and to be different from the two soluble starch synthases (SSI<fnref n="1" /> and SSII) (Macdonald and Preiss, 1985). It is reasonable to think that all these enzymes individually or in complexes are specialized in the biosynthesis of specific granule substructures. Five genetic loci have been shown to be directly involved the granule's structure in maize. These include *su-1* and *su-2* (sugary), *ae* (amylose extender), *du* (dull), and *wx* (waxy) (for a review, see Shannon and Garwood, 1984). The defects have been investigated either on their own or in combination with one another (Creech, 1965). Despite many efforts no clear-cut picture has yet emerged explaining the biosynthesis of the starch granule. As yet only amylose synthesis seems to be partly understood. In fact, waxy mutants characterized by the absence of both amylose and the major granule-bound starch synthase have been described in many higher plants (Weatherwax, 1922; Konishi et al., 1985; Hovenkamp-Hermelink et al., 1987) and more recently in unicellular green algae (Delrue et al., 1992). It is now well established that granule-bound starch synthase is the main polypeptide associated with the starch granule and that this waxy protein is the product of the *wx* gene (Echt and Schwartz, 1981; Schwartz and Echt, 1982; Shure et al., 1983; Van der Leij. et al., 1991; Preiss, 1991; Delrue et al., 1992). Granule-bound starch synthase is thus responsible for amylose biosynthesis *in vivo*. Moreover, granule-bound starch synthase has been shown to be able to add glucose *in vitro* to the amylopectin fraction (Leloir et al., 1961; Baba et al., 1987). We have recently demonstrated that in *Chlamydomonas* at least and perhaps in all plants a substantial portion (20%) of the wild-type amylopectin synthesis is also due to the main granule-bound starch synthase (Delrue et al., 1992). In this paper we report the characterization of low starch mutants defective for the main soluble starch synthase due to mutations in the *ST-3* gene. We show that the mutations all lead to an apparent increase of amylose and considerable modification of the residual amylopectin. The latter displayed an

<footnote>* This work was supported by the Université des Sciences et Technologies de Lille, the Ministère de l'Éducation Nationale and by the Centre National de la Recherche Scientifique (Unité Mixte de Recherche du CNRS 111, Director André Verbert). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver-tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>‡ The first two authors have contributed equally to this work.</footnote>

<footnote>§ To whom correspondence should be addressed. Tel.: 20-43-65-43; Fax: 20-43-65-55.</footnote>

<footnote n="1">¹ The abbreviations used are: SSI, starch synthases; HPAE-PAD, high performance anion-exchange chromatography with pulsed amperometric detection.</footnote>
increase in very short (dp2 to 7) chains and a concomitant
decrease of intermediate size (dp8 to 60) glucan chains. Since
no other scorable defects were detected these results may
suggest that the missing soluble starch synthase is necessary
for the synthesis or maintenance of intermediate size amylo-
pectin chains and thus for that of the branched clusters of
amylopectin. The significance of the short chain presence
with respect to the specificity of amylopectin branching is
discussed.

EXPERIMENTAL PROCEDURES

*Materials*—[U-¹⁴C]Glucose 1-phosphate, [D-glucose-U-¹⁴C]adenosine diphosphate glucose were purchased from Amersham (United Kingdom). ADP-glucose, maize amylopectin, and *Pseudomonas amyloferamosa* isoamylase were from Sigma. Glucose 1-phosphate, rabbit muscle glycogen, and rabbit muscle phosphorylase a were obtained from Boehringer (Mannheim, Germany). DEAE-Trisacryl type M was from IBF Biotechnics (Villeneuve la Garenne, France).

*Chlamydomonas Strains, Growth Conditions, and Media*—The two wild-type strains of *Chlamydomonas reinhardtii* used in this study were 137C (*mt* nit-1 nit-2) and 37 (*mt* pab-2 ac-14). I152, I154, and I39 are independent mutants directly derived from strain 137C by x-ray mutagenesis at 2.58 C Rg⁻¹ (at 2% cell survival) and carry the *st*-3-1 (I152), the *st*-3-2 (I154), and the *st*-3-3 (I39) mutant alleles. B9 and B5 are meiotic recombinant strains of *mt* pab-2 ac-14 genotype carrying, respectively, the *st*-3-1 and *st*-3-2 alleles. Genetic techniques are described in Harris (1989b). Standard media are fully detailed in Harris (1989a), while growth conditions, nitrogen-starved media, and diploid clone selection are described in Ball *et al.* (1990, 1991) and Delrue *et al.* (1992).

*Measures of Starch Levels, Starch Purification, and Spectral Properties of the Iodine-Starch Complex*—A full account of amyloglucosidase assays, starch purification on Percoll gradients, and $\lambda_{max}$ measures can be found in Delrue *et al.* (1992).

*Crude Extract Preparation*—Soluble crude extracts were always prepared from late log phase cells (2.10⁶ cells·ml⁻¹) grown in high salts acetate under continuous light (80 µE·m⁻²·s⁻¹). The cell pellet was suspended to a concentration of 2.10⁶ cells·ml⁻¹ in 50 mM Tris acetate, pH 7.5, 2.5 mM dithiothreitol, 10 mM EDTA and was ruptured by mechanical disruption twice (10,000 pounds/square inch) in a French press. The supernatant cleared by a 15-min 10,000 × g centrifugation was either desalted further by loading 1 ml of the extract (3 mg of protein) on PD-10 (10 ml) Sephadex G-25 columns (Pharmacia LKB Biotechnology (Uppsala, Sweden)) and immediately adjusted with the enzyme assay buffer or was dialyzed against the enzyme assay buffer. Crude extracts were kept at the most 1 h on ice. Proteins were determined according to Lowry *et al.* (1951).

*Enzyme Assays*—Total soluble starch synthase activity was assayed in 0.1-ml final volume of 50 mM glycine NaOH, pH 9, 100 mM (NH₄)₂SO₄, 5 mM β-mercaptoethanol, 5 mM MgCl₂, 0.5 mg·ml⁻¹ bovine serum albumin, 10 mg·ml⁻¹ rabbit liver glycogen, 4 mM ADP-glucose containing 1 nmol of [U-¹⁴C]ADP-glucose (specific activity, 200 µCi·µmol⁻¹). After 15 min of incubation at 30 °C, the reaction was stopped by adding 2 ml of ice-cold ethanol. The resulting precipitate was filtered, rinsed, dried, and counted in a liquid scintillation counter. Citrate-stimulated activity was monitored as above except that 0.5 M citrate was added prior to incubation. Branching enzyme assays were performed by using branching amplification of unprimed phosphorylase α-mediated glucan synthesis (Boyer and Preiss, 1978). The procedure adapted for *Chlamydomonas* is described in Ball *et al.* (1991).

*Soluble Starch Synthase Partial Purification*—All steps were carried out at 4 °C. Fresh crude extracts prepared as above from a total of 10¹⁰ cells (300 mg of protein) were subjected to precipitation with (NH₄)₂SO₄ at 35%. The precipitate was redissolved in 2 ml of 50 mM Tris acetate, pH 7.5, 2.5 mM dithiothreitol, 10 mM EDTA and extensively dialyzed against the same buffer (fraction A). The first supernatant was concentrated by precipitation with (NH₄)₂SO₄ at 65% and redissolved as before (fraction B). Fractions A and B were separately loaded on a DEAE-Trisacryl type M column (2.5-cm inner diameter, 35-cm long) fed with 400 ml of a 0-0.5 M KCl linear gradient. 3.5-ml fractions were collected for immediate analysis.

*Zymograms*—5-10 µg of protein from crude extracts were subjected to separation on a native 7.5% precast polyacrylamide gel (Phast System Pharmacia (Uppsala, Sweden)) for 30-45 min. Unlabeled enzyme buffers for branching enzyme and starch synthase staining were the same as above with the exception of 0.01 mg/ml amylose

which replaced glycogen as a primer for the starch synthase. Gels
were incubated 4 and 12 h, respectively, for starch synthase and
branching enzyme and subsequently stained using a freshly prepared
0.2% I₂ 0.02% KI stock solution.

*Starch Fractionation and Methylation*—Separation of starch fractions on TSK HW-75(S) columns (Merck) was as previously detailed (Delrue *et al.*, 1992). Methylation of total starch and of fractions dialyzed and freeze-dried after TSK HW-75 chromatography was performed according to Paz Parente *et al.* (1985) and adapted to starch analysis (Delrue *et al.*, 1992).

*NMR*—The ¹H¹³C NMR spectra were obtained on a Bruker AM-400 WB spectrometer equipped with a 5-mm mixed ¹H/¹³C probehead, operating in the pulsed Fourier transform mode and controlled by an Aspect 3000 computer with an array processor. The ¹H spectra were obtained with a spectral width of 3000 Hz for 16,384 size domain and time domain data points (pulse width 90° for ¹H = 7.2 µs) The ¹³C NMR experiments were performed with the standard Bruker pulse-program POWGATE with ¹H broadband composite pulse decoupling. The products were analyzed in dimethyl sulfoxide-D6/D₂O, 80:20, at 90 °C (δ-CH₃ of dimethyl sulfoxide = 2.6 parts/million for ¹H and 39.6 parts/million for ¹³C). A 90° pulse (4.8 µs) and a 1.0-s recycle delay were used in each experiment.

*Debranching Analysis*—10 mg of freeze-dried material that was purified by TSK HW-75 chromatography was dissolved by heating (100 °C) in 0.21 ml of dimethyl sulfoxide for 10 min and diluted in 1.7 ml of 55 mM sodium acetate, pH 3.5. The mixture was incubated for 4 h at 45 °C with 3 units of isoamylase and the reaction was stopped by heating (100 °C) for 10 min. After cooling and centrifugation at 10,000 × g for 10 min, the water-soluble material was desalted by an additional chromatographic step on a TSK HW-40(S) column (1.6-inner diameter, 50 cm long) fed by 0.5% (v/v) acetic acid. The pooled polysaccharide fractions were evaporated down to 1 ml and subjected both to TSK HW-50(F) chromatography using a 50-cm long column (inner diameter 1.6 cm) fed by 0.5% (v/v) acetic acid and to HPAE-PAD (Carbo PAC PA-100 column (Dionex), 1 ml/min flow, buffer A: 0.1 M NaOH; buffer B: 0.1 M NaOH, 1 M sodium acetate) (gradient: 0-5 min, 95% A, 5% B (isocratic); 5-17 min, 80% A, 20% B (linear); 17-45 min, 70% A, 30% B (linear). Crude separation of small chains could also be monitored prior to desalting by thin layer chromatography on Silica Gel 60 (Kieselgel 60, Merck) using butanol/acetic acid/H₂O (2:1:1.5, by volume) and orcinol sulfuric staining.

RESULTS

*Selection of High Amylose Mutants*—14,000 colonies that had survived the x-ray treatment were replica plated onto nitrogen-free medium supplied with acetate and incubated in the light. *C. reinhardtii* was previously shown to accumulate large amounts of starch when starved for many different essential elements (Ball *et al.*, 1990). Nitrogen starvation is especially suited since it leads to a pronounced destruction of chlorophylls thus enabling immediate staining by spraying iodine vapors onto Petri dishes. If pigment interference still complicated our observations, the staining was repeated after spontaneous bleaching of the clones. This usually happened in less than 24 h after the first lethal iodine treatment. We were thus able to distinguish three different mutant phenotypes which were clearly distinguishable from the typical dark blue stain due to the interaction between iodine and wild-type starch. In addition to the yellow and red mutants that both appeared at a frequency of 10⁻⁴ (Ball *et al.*, 1991; Delrue *et al.*, 1992), there was a third mutant class that appeared at high frequency (4.10⁻⁴) during our x-ray mutagenesis experiments. These were the so-called olive mutants that accumulated low amounts of starch (from 20 to 40%). The presence of the greenish color led us to suspect that these mutants accumulated a high amylose starch. This seemed to be confirmed by measuring a $\lambda_{max}$ of the iodine polysaccharide complex that was over 600 nm. The three independently selected mutants were then crossed with the wild-type strain 37 and all displayed classical single gene mendelian inheritance while the phenotypes of the vegetative heterozygous diploids showed complete recessiveness of the defects. Segregants of opposite
<figure><img src="image_2.png" /><figcaption>FIG. 1. Separation of amylopectin and amylose on TSK-HW75 chromatography. The optical density (A) was measured for each 3-ml fraction at λmax while λmax is displayed as an unbroken thin line. All samples were loaded on the same column set-up as previously described (Delrue et al. 1992). Both the wild-type haploid 137C strain (panel A) and an heterozygous st-3-1/+ diploid (panel D) can be seen to carry the standard type I (~ fraction 52) and type II (~ fraction 67) amylopectins. Strains I152 (panel B) and I39 (panel C) carrying, respectively, st-3-1 and st-3-3 display a single kind of amylopectin (as far as λmax is concerned) showing size heterogeneity. The amylose population of molecules can be seen from fraction 75 to 105.</figcaption></figure>
mating type bearing the nuclear markers from strain 37 and mutations from strains I152 and I154 were used in standard complementation tests both with the olive mutants and with st-2-1 and st-1-1 carrying strains. Results obtained with vegetative diploids were confirmed by meiotic analysis performed on the very same crosses. No complementation was observed and no recombinants were scored on a total of 96 segregants in each case where both parents crossed were of the olive phenotype. Moreover, the mutations segregated independently from both st-1-1 and st-2-1. Since strains I152, I154, and I39 are all independent mutants, we conclude that they represent three different alleles (st-3-1, st-3-2, and st-3-3, respectively) of a new *Chlamydomonas* locus conditioning both starch structure and amount.

**Structural Characterization of Mutant Starch**—In order to further investigate the structural modifications undergone by the st-3 carrying mutants, we subjected their starch to gel filtration analysis and comparison with the wild-type 137C strain from which they were derived (Fig. 1). As previously described (Delrue *et al.*, 1992), TSK HW-75 chromatography separated the wild-type amylopectin into two fractions characterized by different structural properties. The overall increase in amylose was always significant in the mutants relative to the wild-type controls (the amylose of which can fluctuate between 20 and 40% of total starch). While relative amounts of wild-type peak I and II with respect to amylose are subject to some variation, the structural properties of both amylopectin peaks are remarkably constant. It is obvious that all three mutants retain a single amylopectin species very heterogeneous as far as size is concerned but homogeneous with respect to structure as witnessed by the constance of the $\lambda_{max}$ upon complexation with iodine over the entire size span of the amylopectin. The $\lambda_{max}$ of all residual mutant amylopectin peaks were identical to those measured for wild-type peak II (Table I). The increase in $\lambda_{max}$ suggested the presence of longer chains in the amylopectin. These results seemed to question those obtained by methylation analysis of both, total starch and the purified amylopectin or amylose fractions (Table I). This analysis repeatedly yielded a similar percentage of a-1,6-linked residues in the amylopectin of the I152 mutant strain with respect to both wild-type amylopectin peaks. The proton NMR data also suggest the presence of abundant branches in the amylopectin of that strain (Fig. 4).

**Debranching of Wild-type and Mutant Amylopectin**—The only way to reconcile our methylation results with those obtained by monitoring the iodine polysaccharide interaction would be to suppose the presence of numerous very short (dp < 12) glucans at branch points that would not interfere with the formation of high $\lambda_{max}$ iodine polysaccharide complexes.

<table><caption>TABLE I
Structural properties of amylopectins and amylose purified by TSK HW-75 chromatography</caption><thead><tr><th>Origin</th><th>Q<sup>a</sup></th><th>λ<sub>max</sub><sup>b</sup></th><th>2,3,4,6-Glc<sup>c</sup></th><th>2,3,6-Glc<sup>c</sup></th><th>2,3-Glc<sup>c</sup></th></tr></thead><tbody><tr><td>137C I<sup>b</sup></td><td>65</td><td>550</td><td>0.9</td><td>21</td><td>1</td></tr><tr><td>137C II<sup>b</sup></td><td>10</td><td>580</td><td>0.8</td><td>19</td><td>1</td></tr><tr><td>137C amylose</td><td>25</td><td>620</td><td>0.8</td><td>60</td><td>1</td></tr><tr><td>I152</td><td>55</td><td>590</td><td>0.8</td><td>18</td><td>1</td></tr><tr><td>I152 amylose</td><td>45</td><td>620</td><td>0.8</td><td>60</td><td>1</td></tr></tbody></table>

<sup>a</sup> Q represents the percentage of the total glucose contained in each polysaccharide fraction.

<sup>b</sup> The $\lambda_{max}$ is expressed in nanometers rounded to the nearest decimal.

<sup>c</sup> 2,3,6-Glc, 2,3-Glc, 2,3,4,6-Glc represent the methyl ether derivatives, respectively, of an a(1→4)-linked glucose, an a(1→4)- and a(1→6)-linked glucose, and a glucose in terminal non-reducing position. The amounts were compared taking 2,3-Glc as an arbitrary standard.

<figure><img src="image_3.png" /><figcaption>FIG. 2. HPAE-PAD chromatography of water-soluble debranched glucans from wild-type (137C) and mutant (I 152) TSK-HW75-purified amylopectin. Glucans differing by only 1 Glc residue are clearly separated up to dp25. The dp of some of the chains are given on top of each chromatogram. The figure can be directly read as an histogram of amylopectin chain size since the detection procedure is proportional to the number of molecules and not to the amount of Glc present.</figcaption></figure>

Purified amylopectin from strains I152 and our wild-type reference 137C was subjected to debranching using isoamylase from *P. amyloferamosa*. The soluble linear malto-oligosaccharides were then subjected to HPAE-PAD. The results (Fig. 2) clearly show a very significant decrease of intermediate size glucans (8 to at least 25) in the I152 strain. This defect is accompanied in all cases by the presence of abundant very short glucans (DP2 to 7, with a maximum at 2) which thus resolves the aforementioned paradox. These stubs were also revealed by standard thin layer chromatography on Silica Gel 60 (not shown). While HPAE-PAD proved very useful to investigate the distribution of the debranched products up to 25 glucose residues, the analysis was to be completed by TSK-HW 50 gel filtration which showed that the defect is not limited to glucans in the 8-25 glucose residue size span but also covers the 25 to 50 dp range which together make up the bulk of the intermediate size chains of amylopectin (Fig. 3).

<sup>13</sup>C and Proton NMR Analysis of Standard and Mutant Amylopectins and Amylose—The proton NMR spectra of amylopectin from an st-2-1 carrying strain was compared to that of an st-3-1 carrying mutant and to that of the amylose fractions, all purified by TSK-HW75 chromatography (Fig. 4). The level of branching of the amylopectin was estimated by integration of the same regions of anomeric proton reso-
<figure><img src="image_4.png" /><figcaption>FIG. 3. TSK HW-50 chromatography of water-soluble debranched glucans from wild-type (■) and mutant (▲) TSK-HW75-purified amylopectin. The water-soluble glucans generated by debranching of equal amounts of purified amylopectin were loaded on the same column set up. The dp scale was generated by using maltose and maltoseptaose as external standards and the λ<sub>max</sub> of the debranched glucans as a function of dp as internal standard. Glucose amount were estimated by using the amyloglucosidase assay. A 60% overall decrease could be scored in the amount of glucose in the intermediate size (dp7 to 50) glucan fraction. Glucans of dp > 80 cannot be seen because of their selective retrogradation into insoluble material.</figcaption></figure>

nances of the mono- and disubstituted glucose (Gidley, 1985) (δ ~5.2 and 4.85 parts/million, respectively). Branching was calculated to be on the order of 1–20 for the *st-2-1* standard amylopectin and 1–10 for the *st-3-1* amylopectin confirming thus that the latter is at least as branched. The branching of amylose was undetectable and estimated to be inferior to 1–50 in the same experiment. The ¹³C NMR spectrum of the mutant amylopectin differs drastically from that of our *st-2-1* carrying reference strain. Since this strain contains abundant intermediate size glucans of the amylopectin clusters, we conclude that the signals missing at both the C₁ and C₄ resonance regions reflect a selective decrease of these chains leading to a simplification of the ¹³C NMR spectrum and a relative enhancement of the main signals. On the whole, while the level of branching is at least as high in the *st-3-1* amylopectin, the ¹³C NMR spectrum places this material as intermediate between classical amylose and amylopectin.

*Investigation of the Enzymological Defect in st-3 Carrying Strains*—In order to correlate the defect in intermediate size glucan synthesis with a particular enzyme of starch metabolism, we have assayed ADP-glucose pyrophosphorylase phosphoglucomutase branching enzymes α-amylase soluble and granule-bound starch synthases as well as phosphorylase in crude extracts. A 40–60% decrease in total glycogen-primed soluble starch synthase activity was found as the sole scorable defect in all *st-3* carrying haploid strains. This was confirmed by similar experiments performed in homozygous mutant and wild-type diploids. Interestingly, heterozygous vegetative diploids displayed intermediate activity levels which contrasted with the complete recessiveness displayed by the starch structure phenotype as witnessed by TSK HW-75 chromatography. This suggests that the amount of enzyme activity in wild-type cells is not rate-limiting. These first crude experiments were confirmed by analyzing the activities on native gels where a complete wipe out of all amylose-primed glucan synthesis was detected for all strains carrying *st-3* alleles (*st-3-1*, *st-3-2*, and *st-3-3*). Heterozygous diploids were characterized by intermediate intensities of the band stains on zymograms

thus confirming the crude extract assays. In order to rule out an interference on amplification of the starch synthase-mediated glucan synthesis due to the decrease of a particular branching enzyme activity, we decided to assay these enzymes both in crude extracts and in zymograms. No significant quantitative nor qualitative difference could be scored in the mutants with respect to the wild-type strain. Two very clear bands could be scored on native gels that were unaltered in the mutants.

*Biochemical Characterization of Soluble Starch Synthase Activities in Wild-type and st-3-1 Carrying Strains*—In addition to the lowering of the glycogen-primed starch synthase activity, crude extracts of mutant and wild-type strains could be distinguished by three criteria: $K_m$ for ADP-glucose, stimulation or inhibition (1.4-fold for the mutant, 0.5-fold for the mix of wild-type activities) by 0.5 M citrate in the presence of glycogen, and the presence of a stain on amylose-primed zymograms. This enabled us to follow and separate in wild-type strains two distinct activities by $(NH_4)_2SO_4$ precipitation, one of which (which precipitated at 65% $(NH_4)_2SO_4$ (fraction B)) was clearly related to the activity remaining in the mutant. In order to further characterize the soluble starch synthase activities, the 35% (fraction A) (Fig. 5) and 65% (fraction B) precipitates were subjected separately to DEAE-Trisacryl chromatography using a linear 0–0.5 M KCl gradient. The fraction A activities eluted in two major peaks (SII<sub>a</sub> and SII<sub>b</sub>) that could be essentially distinguished by their $K_m$ for ADP-glucose (Fig. 5 and Table II). Both of these were stable at –20 °C for over 4 weeks and gave clear-cut comigrating bands on amylose-primed zymograms. SII<sub>a</sub> and SII<sub>b</sub> disappeared in fraction A of the mutant to be replaced by a small peak of activity that we have named SII<sub>m</sub>. This residual activity is clearly much more related to the SI of fraction B than to that of both SII<sub>a</sub> and SII<sub>b</sub> (Table II). It does not yield a stain on amylose-primed zymograms and is unstable upon freezing. SII<sub>a</sub> and SII<sub>b</sub> are thus most probably two forms depending on the same *st-3* gene product. Whether these forms are gener-
Amylopectin Defective Mutants of Chlamydomonas

<figure><img src="image_5.png" /><figcaption>FIG. 4. ¹H and ¹³C spectra of purified amylopectin and amylose. Amylose (panel A) and amylopectin (panel B) purified by TSK HW-75 chromatography from strain I152 carrying st-3-1 were compared to our amylopectin reference purified from strain 18B (panel C). The ¹³C resonances of all Glc carbons are displayed and numbered from 1 to 6 while only those proton resonances spectrum that were used to estimate the amount of branching are boxed and shown by arrows.</figcaption></figure>

ated by the purification procedure or are present as such in *vivo* remains to be determined.

Fraction B in the wild-type was characterized by the presence of a unique activity peak eluting earlier in the gradient that we have thus called soluble starch synthase I. This activity is clearly different from both SII<sub>a</sub> and SII<sub>b</sub> and somehow more related to SII<sub>m</sub> and SI<sub>m</sub> (Table II). It does not give a stain on amylose-primed zymograms.

The peak fractions from SII<sub>a</sub> and SII<sub>b</sub> were purified 99- and 83-fold with respect to the total mix of soluble starch synthase activities in the crude extract. SII<sub>m</sub> was purified 106-fold with respect to the crude extract activities. The yields are summarized in Table III.

Fraction B in both strains was essentially devoid of branching enzyme activity. Fraction A-branching enzymes from the mutant and the wild-type eluted in two major peaks at the same position in the gradient with comparable activities (as measured by the phosphorylase assay) suggesting that the st-3-1 mutation does not affect the branching enzyme activities.

DISCUSSION

While a number of models have been proposed for the structure of amylopectin, the main component of the starch granule, all of the recent evidence now favors the so-called cluster models (for a review, see Manners, 1989). Although
the detailed organization of the glucan chains in these models remains controversial, the general features are essentially the same. Intermediate size glucans (dp5 to 50) are packed together in highly ordered clusters that are linked to one another by much longer chains. These clusters make up the bulk of the amylopectin fraction. The relatively small chain lengths of the packed glucans also explain part of the interaction of amylopectin with iodine. The development of color is known to start with maltododecaosis at 500 nm. The $\lambda_{max}$ progressively increases up to 550 nm for 40 glucoside residues, the general color being purple. The rich blue tone which typifies starch starts developing above 50 glucoside residues (Thorn and Mohazzeb, 1990). The abundance of the intermediate size

chains explains the $\lambda_{max}$ of both amylopectin peak I (555 nm) and waxy amylopectin (540 nm). However, peak II (580 nm) and the amylopectin of the st-3 mutations (ranging from 575 to 595 nm depending on the st-3 allele present) are seemingly impoverished of standard branched clusters. This was largely confirmed by our debranching analysis. The presence of numerous very short chains explains neatly our methylation results. It is very tempting at this stage to infer that these branched stubs are the cluster roots that were not elongated by a defective starch synthase. However, the lengths of the chains are smaller than what one would expect if they were the direct products of the branching reaction which produces chains ranging from dp ~8 to over 200 (Takeda et al., 1993). This holds of course if this reaction turns out to be similar to that described for higher plants. One can also speculate that a soluble starch synthase is needed to resynthesize chains that have been recessed by starch phosphorylase.

High amylose mutants have been scored in maize, barley, rice, and peas. In the latter, it has been recently shown that the *r* (rugosus) locus that was identified by Mendel is the structural gene of one of the pea's branching enzyme (Bhat-tacharyva et al., 1990). Wrinkled peas display a 30% reduction in starch and a 2-fold increase in amylose content. The little amylopectin left does not seem to be much modified. However, wrinkled peas accumulate a novel fraction known as intermediate material which seems smaller than amylose but more branched. They are thus not close to the phenotype of our st-3 carrying mutants. An analogous situation has been described in *ae* maize (amylose extender) which displays a phenotype a little closer to our st-3 carrying mutants. In that well documented case, an overall 10-20 nm increase in $\lambda_{max}$ of the amylopectin fraction was indeed reported (Yeh et al., 1981) that was correlated with a 2-fold increase in amylose content and a substantial reduction in total starch synthesis. However, the amylose extender phenotype is not due to a defect in starch synthase but as in the previous case due to a mutation of what appears to be the structural gene of maize branching enzyme II (Boyer and Preiss, 1978; Boyer et al., 1980; Preiss and Boyer, 1980; Singh and Preiss, 1985). Despite many efforts we found no defects in branching enzyme activities whether in crude extracts, in zymograms, or in partially purified extracts. In addition the number of $\alpha$-1,6 linkages has not decreased the slightest, on the contrary, in st-3-1 carrying strains. It is obvious that all st-3 carrying mutants are specifically defective for a subset of soluble starch synthase activities that can be compared to higher plant-soluble starch synthase II. In addition the activity responds proportionally to the number of wild-type alleles in vegetative dip-

<figure><img src="image_6.png" /><figcaption>FIG. 5. Chromatography of mutant- and wild-type-soluble starch synthases on DEAE-Trisacryl. Wild-type strain 137C (panel A) and mutant I152 (panel B) glycogen-primed soluble starch synthase activities recovered in fraction A (see text) were detected in the presence (▲) or absence (■) of 0.5 M citrate. Starch synthase activities are expressed as nanomole of ADP-glucose incorporated into glycogen per milligram protein and per minute. Branching enzyme activities are plotted as dots on the assayed fractions and are expressed as micromoles of glucose incorporated/minute and per milligram of protein into glucan using the phosphorylase assay.</figcaption></figure>

<table><caption>TABLE II
Soluble starch synthase properties from the wild-type (137C) and mutant (I152) strains</caption><thead><tr><th>Substrate</th><th>SIa (137C)<br>35%</th><th>SIb (137C)<br>35%</th><th>SI (137C)<br>65%</th><th>SI<sub>m</sub> (I152)<br>35%</th><th>SI<sub>m</sub> (I152)<br>65%</th></tr><tr><td></td><td colspan="5" style="text-align:center;">K<sub>m</sub> values<sup>a</sup></td></tr></thead><tbody><tr><td>ADP-glucose</td><td>0.29 ± 0.1</td><td>0.06 ± 0.01</td><td>1.1 ± 0.6</td><td>0.9 ± 0.3</td><td>1.8 ± 0.6</td></tr><tr><td>Amylopectin</td><td>0.5 ± 0.1</td><td>0.25 ± 0.1</td><td>0.28 ± 0.1</td><td>0.2 ± 0.1</td><td>ND<sup>b</sup></td></tr><tr><td>Amylopectin + 0.5 M citrate</td><td>0.019 ± 0.005</td><td>&lt;0.005</td><td>&lt;0.003</td><td>&lt;0.03</td><td>ND</td></tr><tr><td colspan="6"><strong>Relative activities to amylopectin<sup>c</sup></strong></td></tr><tr><td>Amylopectin</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>Glycogen</td><td>1.9</td><td>0.87</td><td>1.86</td><td>1.1</td><td>1.6</td></tr><tr><td>Amylopectin + 0.5 M citrate</td><td>1.3</td><td>0.67</td><td>1.88</td><td>ND</td><td>2.3</td></tr><tr><td>Glycogen + 0.5 M citrate</td><td>0.36</td><td>0.14</td><td>3.4</td><td>1.97</td><td>4.5</td></tr><tr><td>0.5 M citrate alone</td><td>0.3</td><td>0.12</td><td>0.1</td><td>0.1</td><td>0.1</td></tr><tr><td>0.1 M maltotriose</td><td>&lt;0.04</td><td>&lt;0.04</td><td>0.26</td><td>&lt;0.06</td><td>ND</td></tr></tbody></table>

<sup>a</sup> K<sub>m</sub> constants expressed in millimolar (ADP-glucose) or milligrams/milliliter (amylopectin and glycogen) are given as mean ± S.E. from three separate experiments originating from a single purification.

<sup>b</sup> ND, not determined.

<sup>c</sup> Amylopectin and glycogen concentrations were adjusted to 5 mg/ml.
<table><caption>TABLE III
Partial purification of wild-type (137C) and mutant (1152) soluble starch synthases from Chlamydomonas</caption><thead><tr><th rowspan="2">Strain step</th><th colspan="2">Volume</th><th rowspan="2">Protein</th><th rowspan="2">Specific activity<sup>a</sup></th><th colspan="2">Purification</th></tr><tr><th>ml</th><th>mg</th><th>-fold</th><th>%</th></tr></thead><tbody><tr><td>137C Crude extract</td><td>24</td><td>90</td><td>6.25</td><td>1</td><td>100</td><td></td></tr><tr><td>137C Fraction A</td><td>12.5</td><td>9.2</td><td>39.5</td><td>6.4</td><td>34</td><td></td></tr><tr><td>137C SIIa</td><td>3.5</td><td>0.1</td><td>621</td><td>99</td><td>11</td><td></td></tr><tr><td>137C SIIb</td><td>3.5</td><td>0.06</td><td>519</td><td>83</td><td>5.5</td><td></td></tr><tr><td>I152 Crude extract</td><td>20</td><td>64</td><td>0.9</td><td>1</td><td>100</td><td></td></tr><tr><td>I152 Fraction A</td><td>10</td><td>12</td><td>4.03</td><td>4.5</td><td>84</td><td></td></tr><tr><td>I152 SIIm</td><td>3.5</td><td>0.08</td><td>95.7</td><td>106</td><td>13.3</td><td></td></tr></tbody></table>

<sup>a</sup>Specific activity is expressed as nanomoles of ADP-glucose incorporated into glycogen per minute and per milligram of protein. All calculations were performed on the peak fractions.

loids, suggesting that ST-3 is the structural gene for SSII. It could also be that of at least one of the subunits of this enzyme if the protein in *Chlamydomonas* happens to be multimeric as is the case for the *S. cerevisiae* glycogen synthases. In yeast glycogen synthase is a 310-kDa heterotetramer made of two 85- and 77-kDa subunits encoded by *GSY1* and *GSY2*, respectively (Peng *et al.*, 1990; Farkas *et al.*, 1990, 1991). Both of these can function on their own in strains carrying disruptions for either *GSY1* or *GSY2*. Our partial purification results could also be easily interpreted along similar lines.

The only other mutant that has been shown to be defective for a soluble starch synthase is the maize *du* (dull) mutant. This mutant that displays as *ae* a 30% reduction in starch accumulation and a net increase in amylose content was not reported to have an increase in the $\lambda_{max}$ of the amylopectin iodine complex (Yeh *et al.*, 1981). Debranching and gel filtration of dull amylopectin yielded a profile that was close but not identical to wild-type, having a small but detectable increase of very short glucans and a slight decrease of intermediate size chains (Inouchi *et al.*, 1987). The picture is further complicated by the fact that both SSII and branching enzyme II are defective in that mutant (Boyer and Preiss, 1978; Preiss and Boyer, 1980). For this reason it was inferred that *du* is a regulatory gene affecting both enzymes. However, branching enzymes and starch synthases may act as complexes in the biosynthesis of starch and *du* might very well be the structural gene of one of the subunits of such a complex. Finally, the presence of the *su-2* mutation in maize has been very recently shown (Takeda and Preiss, 1993) to yield an increase in both amylose content and $\lambda_{max}$ of the amylopectin iodine complex. In this case the amylopectin also displayed the increase in very short chains and the relative decrease in intermediate size glucans. The enzymological defect due to *su-2* remains at present unknown.

It is generally believed, and rightly so, that the branching enzymes are essential in determining the specificity of starch versus glycogen synthesis. This is confirmed *in vivo* by the genetic and molecular analysis of both the *pea r* and the maize *ae* loci. Furthermore, it has been recently shown that the potato-branching enzyme gene complements mutations in the *Escherichia coli*-branching enzyme structural gene (Kossmann *et al.*, 1991). The polysaccharide accumulated by *E. coli* under these circumstances shares some of plant amylopectin properties nailing down on a molecular basis that the *E. coli* glycogen synthase merely assists the potato enzyme in the synthesis of an amylopectin-like molecule. Thus, one would be tempted to think that the specificity of amylopectin synthesis relies solely on the branching enzymes. The evidence presented above suggests that the soluble starch synthase

defect is presently the only scorable enzyme deficiency in st-3 carrying strains of *Chlamydomonas*. We must admit though that further experiments are necessary in order to rule out subtle but nevertheless important branching enzyme activity alterations. Taken together with our recent finding that granule-bound starch synthase controls the synthesis of a substantial portion of amylopectin, the results suggest that in the plant cell any starch synthase is not just polymerizing glucose under the structural dictate of the branching enzymes. The specificity of the structure is equally dependent on the very nature of the starch synthases, probably both by their ability or inability to synthesize glucans of particular length and also to work as complexes with particular branching enzymes.

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