1 STARCH SYNTHASE 4 Domain Functions 2 3 4€¦ · l., 2013; Ragel et al., 2013). These findings are . 100. consistent with a role for SS4 upstream of the other SSs, i.e., a role

1

STARCH SYNTHASE 4 Domain Functions 1

2

Corresponding Author: 3

Samuel C Zeeman 4

Institute of Molecular Plant Biology 5

Department of Biology 6

ETH Zurich 7

CH-8092 Zurich 8

Switzerland. 9

Email: [email protected] 10

Telephone: +41 44 632 8275 11

12

Plant Physiology Preview. Published on November 13, 2017, as DOI:10.1104/pp.17.01008

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2

Distinct Functions of STARCH SYNTHASE 4 Domains in Starch Granule 13

Formation1 14

Kuan-Jen Lu2, Barbara Pfister, Camilla Jenny, Simona Eicke, and Samuel C. Zeeman* 15

Department of Biology, ETH Zurich, 8092 Zurich, Switzerland. 16

ORCID IDs: 0000-0001-5401-7500 (K.-J.L.); 0000-0002-4183-9625 (B.P.); 0000-0003- 17

4180-2440 (S.E.); 0000-0002-2791-0915 (S.C.Z) 18

One-sentence summary: In Arabidopsis leaves, the catalytic C-terminal region of 19

STARCH SYNTHASE 4 promotes starch granule initiation while its non-catalytic N-20

terminal region determines starch granules morphology. 21

1This work was funded by the Swiss National Science Foundation (grant no. 31003A– 22

153144/1 to S.C.Z.) and by ETH Zurich. 23

2Present address: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 24

*Address correspondence to: [email protected] 25

The author responsible for distribution of materials integral to the findings presented in 26

this article in accordance with the policy described in the Instructions for Authors 27

(www.plantphysiol.org) is: Samuel C Zeeman ([email protected]). 28

KL and SCZ conceived and designed the experiments. KL, BP, CJ and SE performed the 29

experiments. KL, BP, and SCZ wrote the paper. All authors analysed the results and 30

approved the final version of the manuscript 31

32



3

SUMMARY 33

The formation of normal starch granules in Arabidopsis leaf chloroplasts requires 34

STARCH SYNTHASE 4 (SS4). In plants lacking SS4, chloroplasts typically produce 35

only one round granule rather than multiple lenticular granules. The mechanisms by 36

which SS4 determines granule number and morphology are not understood. The N-37

terminal region of SS4 is unique among SS isoforms and contains several long coiled-coil 38

motifs, typically implicated in protein-protein interactions. The C-terminal region 39

contains the catalytic glucosyltransferase domains, which are widely conserved in plant 40

SS and bacterial glycogen synthase (GS) isoforms. We investigated the specific roles of 41

the N- and C- terminal regions of SS4 by expressing truncated versions of SS4 and a 42

fusion between the N-terminal region of SS4 and GS in the Arabidopsis ss4 mutant. 43

Expression of the N-terminal region of SS4 alone did not alter the ss4 mutant phenotype. 44

Expression of the C-terminal region of SS4 alone increased granule initiation, but did not 45

rescue their aberrant round morphology. Expression of a self-priming GS from 46

Agrobacterium tumefaciens also increased the number of round granules. Remarkably, 47

fusion of the N-terminal region of SS4 to A. tumefaciens GS restored the development of 48

wild-type-like lenticular starch granules. Interestingly, the N-terminal region of SS4 alone 49

or when fused to GS conferred a patchy sub-chloroplastic localization similar to that of 50

the full-length SS4 protein. Considered together, these data suggest that, while the 51

glucosyltransferase activity of SS4 is important for granule initiation, the N-terminal part 52

of SS4 serves to establish the correct granule morphology by properly localizing this 53

activity. 54

55



4

INTRODUCTION 56

Leaf chloroplasts typically contain multiple lenticular starch granules that form between 57

the thylakoid membranes. These semi-crystalline, insoluble granules are composed of two 58

glucose polymers - 70-90% amylopectin and 10-30% amylose. Amylopectin is a large 59

branched polymer, where glucose residues are linked via α-1,4-glucosidic bonds to form 60

linear chains that are connected via α-1,6-bonds (branch points). The chains in 61

amylopectin arrange in a distinct tree-like architecture in which unbranched chain 62

segments cluster. Neighboring unbranched chain segments intertwine to form double 63

helices that further align in layers to produce a semi-crystalline matrix (Pérez and Bertoft, 64

2010; Buléon et al., 1998). The minor component, amylose, consists of lightly-branched 65

α-1,4-linked chains and is localized in the amorphous parts of the granule between the 66

crystalline layers formed by amylopectin. 67

Starch is synthesized by three types of enzyme activity – starch synthases (SSs), starch 68

branching enzymes, and isoamylase-type debranching enzymes (Myers et al., 2000; 69

Zeeman et al., 2010; Pfister and Zeeman, 2016). SS transfers the glucosyl moiety from 70

ADP-glucose to the non-reducing end of an acceptor glucan. Branching enzyme transfers 71

part of a linear glucan chain to another chain, forming an α-1,6-linkage. Subsequently, 72

isoamylase removes a fraction of the α-1,6-linkages and is thereby thought to facilitate the 73

formation of the crystalline amylopectin layers (Ball et al., 1996). Most plants contain 74

multiple SS isoforms. These can be separated into six classes based on amino acid 75

sequence: SS1, SS2, SS3, SS4, SS5 and the granule-bound starch synthase GBSS 76

(Nougué et al., 2014). Insight into the roles of each class during starch synthesis has been 77

obtained by studying mutant plants deficient in each isoform, as well as by characterizing 78

the enzymes in vitro. The exception is SS5, which has not been characterized to date. 79

According to these studies, SS1, SS2 and SS3 are important for establishing proper 80

amylopectin structure: SS1 preferentially elongates newly placed branches to a length of 81

around 10 glucose units, and SS2 further elongates these chains to around 18 glucose units 82

(Delvallé et al., 2005; Fujita et al., 2006; Umemoto et al., 1999; Pfister et al., 2014). SS3 83

is proposed to synthesize long, cluster-spanning amylopectin chains (Fontaine et al., 1993; 84



5

Fujita et al., 2007; Pfister and Zeeman, 2016). GBSS synthesizes amylose within the 85

granular matrix (van de Wal et al., 1998; Tatge et al., 1999; Zeeman et al., 2002). 86

In contrast to the other SSs, SS4 does not have a major influence on the structure of 87

amylopectin or on amylose synthesis (Roldán et al., 2007; Szydlowski et al., 2009), 88

although in a heterologous, yeast system it is capable of creating branched glucans 89

together with the BEs and ISA (Pfister et al., 2016). Rather, it appears to play a critical 90

role in starch granule initiation: the Arabidopsis ss4 mutant usually has zero, one, or rarely 91

two or three granules per chloroplast (Roldán et al., 2007; Malinova et al., 2017), instead 92

of five to seven granules found in most wild-type chloroplasts (Crumpton-Taylor et al., 93

2012). The total starch content at dusk is lower in this mutant, yet this starch is 94

incompletely metabolized at night. In young leaves, the phenotype is particularly severe, 95

where most chloroplasts are starch-free (Crumpton-Taylor et al., 2013). The ss4 mutant 96

also accumulates very high levels of ADP-glucose, indicating that the remaining SS 97

isoforms cannot utilize it when SS4 is absent, presumably due to the lack of glucan 98

substrates (Crumpton-Taylor et al., 2013; Ragel et al., 2013). These findings are 99

consistent with a role for SS4 upstream of the other SSs, i.e., a role in granule initiation. 100

Interestingly, the ss3ss4 double mutant is almost completely starch-free, suggesting that 101

SS3 is important for the less frequent initiations that still occur when SS4 is absent 102

(Szydlowski et al., 2009; Seung et al., 2016). In addition to altering the number of starch 103

granules, SS4 appears also to influence the granule’s size and shape: those from leaves of 104

the Arabidopsis ss4 mutant are enlarged and near-spherical instead of lenticular (Roldán et 105

al., 2007; Crumpton-Taylor et al., 2013). 106

The molecular features that give each SS its specific role are largely unknown. All share a 107

common catalytic domain comprised of a glycosyltransferase 5 (GT5) and a 108

glycosyltransferase 1 (GT1) sub-domain (Pfister and Zeeman, 2016). This structure is 109

conserved in prokaryotic glycogen synthases (GS), such as GlgA from Agrobacterium 110

tumefaciens. In addition, most plant SS isoforms have N-terminal extensions of different 111

lengths that, in some cases, have demonstrated functions. For instance, the carbohydrate 112

binding modules in the N-terminal extensions of SS3 help confer a higher affinity for – 113

and activity on - its glucan substrates (Senoura et al., 2007; Valdez et al., 2008). 114



6

Several SSs have been shown to engage in protein-protein interactions, often with other 115

starch biosynthetic enzymes (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008, 2009, 116

Liu et al., 2009, 2012). For example, an external helix on the catalytic domain of GBSS 117

enables it to engage in a coiled-coil-mediated interaction with PROTEIN TARGETING 118

TO STARCH (PTST), which helps localize GBSS to the starch granule (Seung et al., 119

2015). Similarly, SS4 was recently shown to bind to PTST2 - a protein related to PTST 120

which also contains a carbohydrate-binding module and predicted coiled-coil motifs 121

(Seung et al., 2017). This interaction appeared to involve the catalytic domain of SS4, 122

although the exact interaction site between SS4 and PTST2 has not yet been identified. 123

Like ss4 mutants, the ptst2 mutants have reduced numbers of starch granules, and this 124

phenotype was exacerbated by loss of another homolog (PTST3; Seung et al., 2017). 125

PTST2 was proposed to bind long malto-oligosaccharides with its carbohydrate-binding 126

module and provide them as substrates to SS4 (Seung et al., 2017). This idea is broadly 127

consistent with other observations that mutations affecting malto-oligosaccharide 128

metabolism affect starch granule initiation (Malinova et al., 2017; Satoh et al., 2008; 129

Seung et al., 2016). 130

The unique N-terminal region of SS4 is predicted to contain coiled-coil motifs (Leterrier 131

et al., 2008), although this region is not needed for interaction with PTST2. However, 132

fluorescent tagging showed that the N-terminal region confers a sub-chloroplastic 133

localization to SS4, possibly at areas peripheral to starch granules (Gámez-Arjona et al., 134

2014). The N-terminal region of SS4 was also reported to enable its association with 135

thylakoid membranes and to allow interaction with the thylakoid- and plastoglobule-136

bound fibrillin proteins FBN1a and FBN1b (Gámez-Arjona et al., 2014). It is unclear 137

whether this a coiled-coil-mediated interaction. Further, the significance of this interaction 138

remains to be established since mutants deficient in both FBN1a and FBN1b have a 139

normal starch phenotype. 140

Despite excellent progress in defining the role of SS4, the mechanisms by which it 141

regulates the initiation of starch granules and controls their morphology are not fully 142

understood. SS4 is presumed to determine the number of starch granules in a chloroplast 143

by synthesizing specific amylopectin precursors (Roldán et al., 2007; Szydlowski et al., 144



7

2009) – a role facilitated by its interaction with PTST2 (Seung et al., 2017). This 145

emerging model contrasts with the priming of glycogen synthesis in other organisms. In 146

metazoans and fungi, glycogen synthesis is primed by glycogenin proteins, which self-147

glucosylate to create primers that GS and branching enzymes can elaborate (Wilson et al., 148

2010; Ball et al., 2011). In prokaryotes, the priming process is less well studied. In A. 149

tumefaciens, there is evidence that the GS can self-glucosylate when supplied only with 150

ADP-glucose, thereby synthesizing glucan primers and fulfilling an analogous role to the 151

eukaryotic glycogenins (Ugalde et al., 2003). 152

Previously, we showed that expression of A. tumefaciens GS in Arabidopsis ss3ss4 153

mutants could overcome the defect in starch granule initiation (Crumpton-Taylor et al., 154

2013), though granule morphology was still aberrant. To extend the understanding of how 155

SS4 contributes to starch granule formation in vivo, we investigated the role of its N- and 156

C-terminal regions by expressing truncated forms in the ss4 mutant background. We 157

analyzed the localization of these truncated forms within the chloroplast and their capacity 158

to complement the mutant phenotype. We further fused the N-terminal part of SS4 to A. 159

tumefaciens GS and compared it to expression of GS alone. We show that the N-terminal 160

region of SS4 alters the localization of GS and allows the formation of normal lenticular-161

shaped starch granules. We discuss the distinct functions of the catalytic C-terminal 162

domain and non-catalytic N-terminus in starch granule initiation and growth. 163

164

RESULTS 165

Generation of ss4 Plants Expressing A. tumefaciens GS or SS4 Variants 166

The A. tumefaciens GS was fused N-terminally to the yellow fluorescent protein (YFP), 167

targeted to the plastids by the transit peptide from the Arabidopsis Rubisco small subunit 168

(At5g38430; amino acid residues 1-53), and expressed in the ss4-1 single mutant (Col-0 169

background) (YFP-GS; Figure 1). Since GS is homologous to the C-terminal half of SS4, 170

we also designed a truncated SS4 construct consisting of its C-terminal region (amino 171

acids 476 to 1040; including both GT1 and GT5 domains and a region immediately 172



8

preceding them that is conserved in SS4 orthologs) that was N-terminally fused to YFP 173

(YFP-SS4C; Figure 1). Further, we generated a construct containing the N-terminal half 174

of SS4 (amino acids 1 to 544, also including the conserved region preceding the GT 175

domains; SS4N-YFP) C-terminally fused to YFP. We also made a chimeric construct 176

where this N-terminus of SS4 was fused to full-length A. tumefaciens GS and YFP (YFP-177

SS4N-GS or SS4N-GS-YFP, depending on the position of the YFP; Figure 1). Finally, 178

constructs encoding the full-length SS4 protein fused to YFP (SS4-YFP) and chloroplast-179

targeted YFP alone served as controls (Figure 1). In all cases, constitutive expression was 180

driven by the CaMV 35S promoter. 181

For each construct, we obtained numerous independent transgenic lines in the ss4 mutant 182

background. We then selected three lines of each with different expression levels, judged 183

by immunoblots on total protein extracts using either a monoclonal anti-YFP antibody or 184

polyclonal antibodies against SS4 (Figure 2A). Every plant line gave a band 185

corresponding to the expected molecular weight of its mature introduced protein (i.e. 186

lacking the chloroplast transit peptide). These bands were absent in protein extracts of the 187

ss4 parent line and the wild type. There were additional, often closely migrating bands 188

apparent in the transformed plant lines (Figure 2A). It is possible that these additional 189

bands reflect post-translationally modified forms of the introduced protein (e.g. due to 190

phosphorylation or proteolysis). In case of YFP-GS, we observed additional slowly 191

migrating bands, which possibly resulted from self-glucosylation of the protein. 192

We investigated whether the chain-elongating activities of introduced enzymes were 193

detectable on zymograms (native PAGE gels supplemented with glycogen as a substrate; 194

Figure 2B). After incubating the gels in an ADP-glucose-containing medium, activity 195

bands were visualized as dark bands by iodine staining, indicating elongation of glycogen 196

chains. Bands corresponding to the endogenous starch synthases SS1 and SS3 (Wattebled 197

et al., 2005; Zhang et al., 2005; Szydlowski et al., 2009) were detected in all plant 198

extracts. In addition, a dark, smeared band appeared in lines expressing YFP-GS. Among 199

these lines, YFP-GS_3 had the strongest GS activity and YFP-GS_1 had the weakest, 200

consistent with the immunoblotting results (Figure 2A). Faint bands were visible in some 201

SS4N-GS-YFP-expressing and YFP-SS4N-GS-expressing lines. No additional activity 202



9

bands were visible in lines expressing YFP-SS4C, SS4N-YFP, or SS4-YFP. However, 203

these zymograms are evidently not able to detect all glucosyl transferase activities, since 204

the endogenous SS2 and SS4 activities are not visible either (Szydlowski et al., 2009; 205

Pfister et al., 2014). Multiple pale bands were also visible in the zymograms. These result 206

from glucan hydrolytic activities that digest the glycogen substrates and could potentially 207

mask the activities of co-migrating glucan synthases. 208

Plant Growth and Leaf Starch Content Are Partially Restored by GS or the C-209

Terminal Region of SS4 210

We evaluated our transformed plant lines for their growth phenotypes and for their leaf 211

starch accumulation by iodine staining (Figure 3). As previously reported, the ss4 mutant 212

grew slowly and had pale and starch-free young leaves (Roldán et al., 2007; Crumpton-213

Taylor et al., 2013). The older leaves stained for starch at the end of day and, unlike the 214

wild type, did not to fully degrade it during the subsequent night. Control ss4 plants 215

expressing chloroplast-targeted YFP alone were indistinguishable from the ss4 parent. 216

The YFP-GS-expressing plants had greener rosette leaves and line 1 displayed 217

significantly improved growth compared with ss4 mutants. Lines YFP-GS_1 and YFP-218

GS_2 also showed complementation in terms of starch accumulation, as both young and 219

mature leaves stained for starch. Yet part of this starch remained after the night, resulting 220

in patchy staining of the leaves. Similarly, transformants expressing the C-terminal region 221

of SS4 (YFP-SS4C lines) grew better than ss4 mutants and accumulated starch in all 222

rosette leaves. These plants also did not fully metabolize their starch during the night. 223

Interestingly, expression of GS fused to the N-terminal region of SS4 (SS4N-224

GSYFP/YFP-SS4N-GS) resulted in improved growth and iodine-staining patterns that 225

were very similar to those of the wild type and SS4-YFP control plants: all leaves were 226

green, accumulated starch during the day and degraded most of it in the subsequent night. 227

In contrast, when the N-terminal region of SS4 was expressed alone (SS4N-YFP plants), 228

the phenotype was indistinguishable from that of the ss4 mutant. Thus, GS or the C-229

terminal region of SS4 alone partially complement the ss4 mutant phenotype. The extent 230



10

of complementation is improved by the presence of the N-terminal region of SS4, but the 231

N-terminal region alone cannot complement the mutant. 232

The Influence of the N-Terminal Region of SS4 on Starch Metabolism 233

Iodine staining is a qualitative rather than quantitative indication of starch content. To 234

quantify the starch content in our lines, we harvested whole rosettes at the end of a normal 235

day and night, digested the starch enzymatically, and measured the released glucose 236

(Figure 4). As expected, ss4 plants accumulated less starch during the day than the wild 237

type. They also degraded only half of their starch by the end of the night. These results 238

were consistent with our observations from iodine staining (Figure 3) and previous reports 239

(Roldán et al., 2007; Crumpton-Taylor et al., 2013). 240

The YFP-GS lines varied considerably in starch content at the end of day, ranging from 241

near-normal amounts of starch in YFP-GS_1 to starch levels even lower than in ss4 242

mutants in case of YFP-GS_3 (Figure 4). Thus, starch content correlated inversely with 243

the abundance and activity of GS (Figure 2). Consistent with the iodine-staining (Figure 244

3), the YFP-GS lines did not fully mobilize their starch during the night. The lines 245

expressing YFP-SS4C reached starch levels close to those of the wild type, but also 246

degraded only ~70% of the starch reserve during the night. 247

By contrast, SS4N-GS and SS4-YFP plants showed near-wild-type starch accumulation 248

and breakdown, degrading around 80-90% of their starch by dawn (Figure 4). Thus, 249

having the N-terminal region of SS4 positively influenced starch turnover both when 250

combined with GS or the C-terminal region of SS4. Yet, when expressed on its own, the 251

N-terminal region of SS4 again showed no effect on the pattern of starch accumulation, as 252

SS4N-YFP plants phenocopied the ss4 mutant (Figure 4). 253

SS4 Domains Influence Starch Granule Shape and Numbers Independently 254

It was proposed that the reduced starch synthesis and degradation rates in ss4 mutants may 255

be a consequence of its deposition as fewer, enlarged and rounded granules when 256

compared to the wild type (Roldán et al., 2007). Since a number of our complementation 257



11

lines also still displayed impaired starch metabolism, we investigated the number and 258

morphology of starch granules within their chloroplasts. Leaf number 7 (an expanding 259

source leaf; Supplemental Figure S1) was harvested at the end of day, and samples from 260

the middle of the leaf lamina were fixed and embedded in resin. We obtained light 261

micrographs (LMs; Figure 5 and Supplemental Figure S2) and transmission electron 262

micrographs (TEMs; Figure 5 and Supplemental Figure S3) of the leaf mesophyll tissue, 263

where the majority of leaf starch is synthesized. To examine granule morphology in more 264

detail, we also purified starch granules from whole rosettes and visualized them by 265

scanning electron microscopy (SEM; Figure 5 and Supplemental Figure S4). 266

In sections from wild-type chloroplasts, we predominantly observed multiple ellipsoid 267

starch granules (Figure 5), consistent with the flattened, lenticular shape of the granules 268

seen by SEM (Figure 5). In contrast, 80% of the sections of the ss4 mutant chloroplasts 269

were devoid of starch (Supplemental Table S1). The minority of chloroplast sections 270

contained starch, usually as one larger and rounded granule, as previously reported 271

(Crumpton-Taylor et al., 2013; Roldán et al., 2007). It should be noted that chloroplast 272

sections of ~750 nm thickness capture only a part of the chloroplast, meaning that the 273

number of starch-containing chloroplasts will be slightly underestimated. 274

The lines expressing YFP-GS differed from the ss4 parental line in that they typically 275

contained multiple rounded granules per chloroplast section (Figure 5; Supplemental 276

Figure S2, Supplemental Figure S3, Supplemental Figure S4), and only a few (10-20%) of 277

the chloroplast sections were starch-free (Supplemental Table S1). Interestingly, 278

approximately 10% of the chloroplast sections contained supernumerary granules, which 279

were tiny and of irregular shape (Figure 6). The proportion of chloroplast sections 280

containing these aberrant granules was highest in the line YFP-GS_3 (Supplemental Table 281

S1), which expressed GS to the highest level (Figure 2). In the YFP-SS4C lines, ~90% of 282

chloroplast sections contained starch granules, similar to the YFP-GS lines. However, 283

granules produced in the YFP-SS4C lines were larger and fewer in number than those 284

from the YFP-GS lines. Interestingly, the granules still exhibited the rounded shape, 285

characteristic of the ss4 mutant (Figure 5; Supplemental Figure S2, Supplemental Figure 286

S3, Supplemental Figure S4). No supernumerary granules were observed in the case of 287



12

YFP-SS4C expression. Thus, while expression of either GS or SS4C in the ss4 288

background facilitated the formation of more starch granules, neither restored the normal 289

lenticular morphology. 290

Expression of the SS4N-GS fusion constructs almost fully complemented the ss4 mutant 291

phenotype: there were multiple granules per chloroplast section and the granules had the 292

wild-type-like shape, despite being slightly smaller. As in the GS-YFP lines, these lines 293

had also contained some chloroplasts with supernumerary tiny granules within a single 294

stromal pocket (observed in ~10-20% of the chloroplast sections). Often, these 295

supernumerary granules had smooth surfaces while in other sections they were irregular in 296

appearance (Figure 7; Supplemental Figure S5). 297

As expected, the three SS4-YFP lines also produced multiple wild-type-like granules per 298

chloroplast section (Figure 5; Supplemental Figure S2, Supplemental Figure S3, 299

Supplemental Figure S4), even though they contained lower amounts of SS4 than the wild 300

type (Figure 2). In this case, no supernumerary granules were observed. Lines expressing 301

SS4N-YFP had starch granules that were indistinguishable from those of the ss4 parent 302

both in terms of morphology and frequency (Figure 5 and Supplemental Table S1), 303

consistent with our other observations that the N-terminal region of SS4 by itself appears 304

to have no effect on starch biosynthesis. 305

We investigated whether the differences in granule morphology were accompanied by 306

changes in amylopectin structure. Therefore, we enzymatically debranched starch from 307

each line and analyzed its chain-length distribution (CLD) by HPAEC-PAD 308

(Supplemental Figure S6). This confirmed that the CLDs of starch from wild-type and ss4 309

mutant plants are very similar (Roldán et al., 2007). The CLDs of starches from our set of 310

transformed lines were also all very similar to that of the wild type. 311

The N-Terminal Region of SS4 Influences the Sub-Chloroplastic Localization 312

Previous work indicated that SS4 is not homogeneously distributed within the chloroplast 313

stroma, but rather localizes to discrete spots at the edges of starch granules. Furthermore, 314

this localization was suggested to be dependent on its N-terminal region (Gámez-Arjona 315



13

et al., 2014; Szydlowski et al., 2009). To investigate the localization of our expressed 316

proteins, we imaged YFP fluorescence in Arabidopsis leaves during the light period by 317

confocal laser scanning microscopy (Figure 8). As expected, free YFP was uniformly 318

distributed within the chloroplast stroma (non-fluorescent patches typically reflect the 319

presence of starch granules). This localization was similar in case of YFP-SS4C, which 320

also gave a uniform stromal YFP fluorescence. This result differs from that described by 321

Gámez-Arjona et al. (2014), where the C-terminal region of SS4 appeared as a single dot 322

at one end of the chloroplast. This is most likely due to differences between stable 323

expression in transformed Arabidopsis lines used here versus transient overexpression N. 324

benthamiana used previously 325

Interestingly, YFP-GS appeared as ring-like structures surrounding the rounded starch 326

granules, indicating that it preferentially associates with the granule surface. When the N-327

terminal part of SS4 was fused to GS, however, it did not uniformly surround the granules 328

anymore, but rather appeared to localize to discrete areas at the periphery of starch 329

granules. This distribution was similar to that of full-length SS4-YFP and the N-terminal 330

region of SS4 alone (Figure 8). Thus, the N-terminal region of SS4 appears both necessary 331

and sufficient to confer the distinct sub-chloroplastic localization typical of SS4. 332

DISCUSSION 333

This work provides significant new insight into the function of SS4 in starch granule 334

formation. On the one hand, the catalytic C-terminal region of SS4 promotes granule 335

formation, presumably via the catalytic activity of its glucosyltransferase domains – a role 336

that can be substituted by A. tumefaciens GS. On the other hand, the N-terminal region of 337

SS4 confers a specific sub-chloroplastic localization, which is critical in determining 338

granule morphology. It is remarkable that this N-terminal region of SS4 similarly guides 339

the activity A. tumefaciens GS, which is then able to substitute surprisingly well for SS4. 340

Agrobacterium GS Increases Granule Number in the ss4 Mutant 341

The data presented here support our previous work indicating that A. tumefaciens GS 342

promotes the initiation of starch granules in the absence of SS4 (Crumpton-Taylor et al., 343



14

2013). In the ss4 parental line, 80% of the chloroplast sections examined had zero 344

granules, with the remainder having one or at most two granules. Most chloroplasts in the 345

GS-expressing lines contained numerous small, round granules. Very few chloroplasts 346

were granule free, while some contained supernumerary granules (Figures 5, Figure 6, 347

Supplemental Table S1). GS could function to increase the number of granules either by 348

increasing the total amount of glucan-elongation activity, such that it increases the 349

frequency of stochastic granule initiations. Alternatively (or in addition), GS could 350

promote granule initiation via its self-priming ability (Ugalde et al., 2003), providing a 351

substrate for the other starch biosynthetic enzymes. Consistent with this idea, 352

immunoblotting and zymogram analyses revealed multiple and/or smeared bands for the 353

YFP-GS protein and its activity, respectively (Figure 2). Such behavior on SDS- and 354

native-PAGE gels was proposed to be due to self-glucosylation, which changes the 355

protein’s molecular weight and properties, thereby affecting its migration (Ugalde et al., 356

2003). 357

Among the GS-expressing lines, granule numbers per chloroplast and appeared to 358

correlate positively with GS protein/activity. However, starch metabolism was far from 359

normal in these lines; granule morphology was still aberrant, as in the ss4 mutant, and the 360

extent of starch metabolism during the diel cycle was highly variable. Surprisingly, starch 361

content at the end of the day was negatively correlated with GS protein/activity: YFP-362

GS_1 had low GS activities and comparable amounts of starch to the wild type while 363

YFP-GS_3 had the largest amount of GS activity but the lowest amount of starch (Figure 364

2, Figure 4). The reason for this is unclear. One possibility is that, despite increasing the 365

number of granule initiations, excessive amounts of GS somehow interfere with the 366

activities of the plant’s endogenous enzymes, restricting the rate of starch biosynthesis. 367

YFP-GS had a distinct localization, surrounding the starch granules (Figure 8). This is 368

somewhat reminiscent of the subcellular localization of the native GS (EcGS) in E. coli 369

cells, which associates with glycogen particles at the cell poles (Wilson et al., 2010). 370

Association of GS with the starch granule surface might crowd-out the other chain 371

elongating, branching and debranching enzymes necessary for starch synthesis. 372

Interestingly, a somewhat analogous phenotype was observed in transgenic potato tubers 373



15

expressing EcGS; these produced less starch, which was more highly branched, and 374

accumulated more soluble sugars (Shewmaker et al., 1994). However, in our lines 375

expressing YFP-GS, chain-length distributions revealed no such alterations in the 376

structure of the remaining starch (Supplemental Figure S6) and no polyglucans were 377

measured in the water-soluble fraction (data not shown). An alternative explanation for 378

the low starch content in the presence of high GS could be that glucan degradation 379

proceeds simultaneously with synthesis. If glucans made by abundant GS together with 380

the other starch biosynthetic enzymes is unable or slow to crystallize, they may be subject 381

to hydrolysis. Such situations have been reported in Arabidopsis (e.g. in the isa1 382

isoamylase-deficient mutants or the ss2ss3 double mutant; Delatte et al., 2005; Pfister et 383

al., 2014), but again correlated with a measurable change in the structure of the starch 384

(Supplemental Figure S6). Interestingly, when GS was expressed in the ss3ss4 mutant 385

background, starch structure was altered, having more short (d.p. 5-7) and fewer 386

intermediate (d.p. 10-15) chains than wild-type starch (Crumpton-Taylor et al., 2013). We 387

suggest that when GS represents a greater fraction of the total synthase activity, its 388

preference for producing short like chains typical of glycogen, becomes more apparent. 389

Despite variation in the amount of starch synthesized, the proportion of starch degraded at 390

night was increased in all three YFP-GS expressing lines relative to ss4 (Figure 4). Low 391

starch turnover in ss4 plants was proposed to result from having fewer, large starch 392

granules - this changes the surface area available for both synthesis during the day and 393

degradation at night (Roldán et al., 2007). Clearly, the numerous small, round granules 394

made in the presence of YFP-GS have a greater surface area on which starch metabolic 395

enzymes can act, compared with the few large, round granules in ss4 plants (Figure 5, 396

Figure 6). Thus, expression of YFP-GS may help proper starch turnover indirectly by 397

increasing the granule number. However, whether the granule surface area is indeed a 398

limiting factor for synthesis and/or degradation remains to be tested. 399

The C-terminal Region of SS4 Alone Promotes Granule Initiation in ss4 Mutants 400

The C-terminal region of SS4 is homologous to the full-length GS from A. tumefaciens 401

and the phenotypes of plants transformed with the SS4C or the GS construct were similar 402



16

in several respects. First, expression of both constructs partially restored starch 403

accumulation, with over 80% of the chloroplasts producing multiple starch granules 404

(Figure 5; Supplemental Figure S2, Supplemental Figure S3). Second, in both cases a 405

greater fraction of the starch could be degraded at night than in ss4 (Figure 4). Third, 406

neither construct altered amylopectin structure compared to the wild type (and ss4 407

parental line; Supplemental Figure S6). However, also in both instances the starch 408

granules still had the rounded ss4-like morphology (Figure 5, Supplemental Figure S4), 409

demonstrating that neither YFP-SS4C nor YFP-GS could rescue all aspects of the ss4 410

mutant phenotype. 411

Despite the similarities between the YFP-SS4C and the YFP-GS lines, the phenotypes 412

were not identical. The YFP-SS4C lines had fewer starch granules per chloroplast than the 413

YFP-GS lines, and we did not observe supernumerary granules (Supplemental Table S1). 414

This might be due to the relatively low amount of YFP-SS4C protein expressed (Figure 415

2A). YFP-SS4C activity could also not readily be detected using zymograms, even though 416

Raynaud et al. (2016) reported that a similarly truncated form of SS4, when 417

recombinantly expressed in E. coli, was measurable this way. Indeed, the phenotypes of 418

the YFP-SS4C lines were most similar to that of YFP-GS_1, i.e. the line expressing the 419

lowest amounts of YFP-GS. It is possible that higher amounts of YFP-SS4C would have 420

led to more granules and lower starch contents, as seen with high levels of YFP-GS 421

expression. 422

However, there were also qualitative differences between the SS4C and GS proteins that 423

could be relevant to the distinct phenotypes observed. First, immunoblots of the YFP-424

SS4C protein did not yield multiple, slow-migrating bands as they did for YFP-GS 425

(Figure 2A). This is presumably because SS4C does not have self-priming activity 426

(Szydlowski et al., 2009) and rather relies on interaction with the recently-described 427

PTST2 and/or PTST3 proteins to deliver its substrates (Seung et al., 2017). Second, our 428

YFP fluorescence data revealed that SS4C was uniformly distributed in chloroplasts, 429

unlike the granule-surface localization of GS (Figure 8), suggesting a lower inherent 430

affinity to glucans. Third, it is possible that the glucan elongating activity and specificity 431



17

of YFP-SS4C differs from that of YFP-GS. Indeed, if glycogen is a poor substrate for 432

SS4C, this might help explain why we did not detect its activity in glycogen-containing 433

native gels. 434

The N-Terminal Domain of SS4 Determines the Morphology of Starch Granules 435

Our data indicate that the N-terminal region of SS4 has a very specific and important role 436

in starch granule biosynthesis. It contains predicted coiled-coil domains between amino 437

acids 204 and 466 (see Pfister and Zeeman, 2016 and references therein). This sequence 438

conserved in SS4 orthologs, but is absent in the other SS isoforms, although some of them 439

have distinct predicted coiled-coil motifs of their own (Pfister and Zeeman, 2016). The N-440

terminal region of SS4 had a patchy sub-chloroplastic localization similar to that of the 441

full-length SS4 protein (Figure 8), suggesting that this region determines the protein’s 442

localization. However, it did not rescue the growth, starch synthesis and turnover, or 443

granule number and morphology phenotypes of ss4 (Figure 3, Figure 4, Figure 5; 444

Supplemental Figures S2-S4). These data show that the N-terminal region of SS4 alone, 445

while correctly localized, is not functional without the catalytic C-terminal domains. 446

Remarkably, both the function and localization of A. tumefaciens GS could be changed by 447

fusing the N-terminal region of SS4 to it. Expressing the chimeric protein (SS4N-GSYFP 448

or YFP-SS4N-GS) in ss4 plants complemented most aspects of the ss4 phenotype: the 449

plants displayed near-normal growth rates (Figure 3), starch levels and nighttime starch 450

degradation (Figure 4). Strikingly, SS4N-GS expression not only increased granule 451

numbers, but also resulted in wild-type-like lenticular starch granules (Figure 5; 452

Supplemental Figures S2-S4). SS4N-GS furthermore showed a sub-chloroplastic 453

localization that resembled that of SS4 rather than that or GS (Figure 8). Nevertheless, we 454

still observed subtle differences to the wild type. For example, there was a weak negative 455

correlation between SS4N-GS abundance and starch synthesis: The line which contained 456

the highest activity of SS4N-GS also produced the lowest amount of starch among the 457

three lines and vice versa (e.g., SS4N-GS-YFP_3 vs. SS4N-GS-YFP_1; Figure 2, Figure 458

4). This correlation was reminiscent of what was observed in lines expressing just GS 459

(discussed above). Moreover, the SS4N-GS-expressing lines had noticeably more starch 460



18

granules than the SS4-complementation lines (Figure 5; Supplemental Figures S2-S3), 461

and sometimes produced supernumerary smooth-surfaced tiny granules within single 462

stromal pockets of chloroplasts - something not seen in the wild type or in SS4-YFP lines 463

(Figure 7; Supplemental Figure S5). 464

The Functions of SS4 465

Granule initiation probably requires more than just priming of polysaccharide synthesis: It 466

may require that the pre-crystalline glucan is not subject to degradation (e.g. by α-amylase 467

or isoamylase; Seung et al., 2016; Pfister et al., 2016), and also that the polysaccharide 468

can be further elaborated and assembled with other glucan molecules to provide the basis 469

for a semi-crystalline starch granule. Potentially, these processes are mediated by protein 470

scaffolds that localize SS4 at distinct sub-plastidial spots. Such a protein scaffold may also 471

serve to guide the biosynthetic machinery after initiation has occurred, directing the 472

pattern of synthesis. Based on ours and other results, we propose that the domain structure 473

of SS4 serves distinct important roles in these aspects of starch granule initiation and 474

growth. 475

The C-terminal domains confer the catalytic activity for glucan chain elongation by SS4 476

(Szydlowski et al., 2009), which may be particularly important during the early steps of 477

granule initiation. The C-terminal region also appears to confer the capacity to interact 478

with PTST2, which provides it with long malto-oligosaccharide substrates (Seung et al., 479

2017). A conserved domain immediately adjacent to the C-terminal glucosyltransferase 480

domains allows SS4 dimerization, which was suggested to be required for activity 481

(Raynaud et al., 2016). Dimerization may promote the elongation of long malto-482

oligosaccharides in proximity to one another. This could increase the likelihood of glucan 483

interactions that must precede the formation of the higher-order, semi-crystalline 484

structures observed in starch granules. In line with these hypotheses, recombinant barley 485

SS4 exhibits significant glucan-elongating activity only on linear malto-oligosaccharides, 486

but not on branched polysaccharides (Cuesta-Seijo et al., 2016). Although the in-vivo 487

substrate affinity by SS4 may be modulated by PTST2, it is tempting to speculate that this 488



19

combination of catalytic specificity, interaction with PTST2 and self-dimerization renders 489

SS4 capable of granule initiation. 490

Notably, barley isoforms of SS1, SS2, SS3 and GBSS did not display such a preference 491

for malto-oligosaccharides, and were equally or more active on branched polysaccharides 492

as on linear oligosaccharides (Cuesta-Seijo et al., 2016). Phylogenetic analyses of starch 493

synthases suggest that SS1 and SS2 are derived from the duplication of an ancient 494

cyanobacterial GBSS gene, while SS3 and SS4 belong to clade that either derived from the 495

host or a chlamydial pathogen (Ball et al., 2011). This difference in origin could mean that 496

SS1 and SS2 do not share the catalytic capacities required for priming with SS4. Indeed, 497

when expressed in yeast, Arabidopsis SS1 and – to a lesser extent – SS2 were less 498

efficient in glucan synthesis than SS3 and SS4 when the yeast’s endogenous glycogenins 499

were absent (Pfister et al., 2016). It therefore appears unlikely that the catalytic regions of 500

SS1 or SS2 alone or fused to the N-terminal region of SS4 would be capable of granule 501

initiation in the ss4 mutant background. 502

The N-terminal region positions SS4 into specific sub-chloroplastic domains. Thereby, it 503

may help define not only where a granule forms, but also the way in which the granule, 504

once initiated, continues to grow, resulting in their characteristic lenticular shape. The 505

nature of these sub-chloroplastic domains and how the N-terminal region localizes to them 506

remain unclear. It seems likely that the N-terminal region exerts its function by interacting 507

with other proteins (i.e. the protein scaffold hypothesized above). Two candidate 508

interactors of SS4 - the thylakoid-associated fibrillin proteins, FBN1a and FBN1b - have 509

already been identified. Yet, these alone cannot explain the localization and function of 510

the SS4 N-terminus, since lenticular granules are still made when both fibrillins are 511

abolished (Gámez-Arjona et al., 2014). Further studies will be needed to identify 512

additional factors and to build a detailed model of the cell biological context for starch 513

granule biosynthesis. 514

Finally, the experiments described here also further highlight the apparent link between 515

granule numbers/morphology and diel starch turnover. The ability of the C-terminal 516

region of SS4 or full-length GS to increase granule numbers, and of the attached N-517



20

terminal region of SS4 to further restore their normal morphology, correlated with 518

increases in starch amount and its diel turnover. This is important since starch metabolism 519

is central to the plant’s ability to control its overall carbon budget and to maintain 520

efficient growth (Stitt and Zeeman, 2012). However, it is important to note that in other, 521

recently described genotypes, this link seems to be broken: plants deficient in or 522

overexpressing the SS4-interacting protein PTST2 have fewer or more numerous granules 523

than the wild type, respectively, yet still maintain near-normal diel starch turnover (Seung 524

et al., 2017). 525

MATERIAL AND METHODS 526

Plant Material and Growth Conditions 527

Arabidopsis wild type Columbia (Col-0), T-DNA insertion mutant ss4-1 (GABI- 528

Kat_290D11), and T2 generation of ss4-1 plants transformed with each of YFP-GS, YFP-529

SS4C, SS4N-GS-YFP, YFP-SS4N-GS, and SS4-YFP were used. Seeds were sown on soil 530

and placed for 2 days at 4°C in the dark, and were grown in Percival growth chamber 531

(CLF Plant Climatics) under 12-h day/12-h night regime. The temperature was 20°C, 532

relative humidity was 70%, and the light intensity was 150 µmoles quanta m-2

s-1

. 533

Cloning and Isolation of Transgenic Lines 534

The full length coding sequence of GS from Agrobacterium tumefaciens (Atu4075) was 535

amplified from genomic DNA using GS-fw and GS-rev primers, given in Supplemental 536

Table S2. The PCR product was cloned into the pDONR221 vector, and was further 537

cloned into the pB7WGY2 vector (modified to contain a chloroplast transit peptide [rCT: 538

amino acids 1-53 of At5g38430] upstream and in frame with YFP) via Gateway™ 539

recombination cloning technology (Invitrogen). The resulting construct was named YFP-540

GS (Figure 1). The same YFP-GS construct as used in Crumpton-Taylor et al. (2013) was 541

introduced into ss4 mutant plants, yielding the two independent lines YFP-GS_1 and 542

YFP-GS_3. The third independent line YFP-GS_2 was generated by crossing the ss4 543

mutant with one of our previously generated YFP-GS lines in the ss3 ss4 background 544

(GS-5-3; Crumpton-Taylor et al. 2013) and selecting for the homozygous YFP-GS, ss4 545



21

and wild-type SS3 alleles. Similarly, the C-terminal domain of Arabidopsis SS4 (SS4C; 546

amino acid residues 467 to 1040) was amplified from the pDONR-AtSS4 plasmid (Roldán 547

et al., 2007) using SS4C-fw and SS4C-rev primers (Supplemental Table S2). The 548

amplified SS4C fragment was cloned into rCT containing pB7WGY2,0 vector and the 549

resulting construct was named YFP-SS4C (Figure 1). The N-terminal domain of SS4 550

(SS4N; amino acid residues 1 to 544) was amplified with attB sites using SS4-fw, SS4N-551

GS-rev primers. The SS4NΔctp-fw and SS4N-GS-rev primers were used to amplify the N-552

terminal domain of SS4 without its predicted chloroplastic transit peptide (amino acid 553

residues 43 to 544). The pDONR-AtSS4 plasmids were used as template. The amplicons 554

of SS4N and GS were cleaved and ligated via Bcl1 restriction sites, cloned into 555

pB7YWG2,0 and into the rCT-containing pB7WGY2,0 vectors. The resulting constructs 556

were named SS4N-GS-YFP and YFP-SS4N-GS (Figure 1). The full-length cDNA of SS4 557

was cloned into pB7WGY2,0 via the Gateway™ LR reaction, and named SS4-YFP 558

(Figure 1). 559

We transformed each expression construct (YFP-GS, YFP-SS4C, SS4N-GS-YFP, YFP-560

SS4N-GS, and SS4-YFP) into ss4-1 mutants. The T1 seeds from each transformation were 561

harvested and sown on soil. Multiple T1 transgenic plants for each transformation were 562

isolated by Basta® resistance screening. Two or three independent lines for each 563

transformation were selected for further analysis, based on different abundances and 564

fluorescence appearance of transformed proteins. T2 plants carrying the ss4-1 allele and 565

transformed constructs were identified by PCR-based genotyping using primers shown in 566

Supplemental Table S2. 567

Immunoblotting and Zymograms 568

Whole rosette from each of 25-day old plants was harvested at the end of day, weighed, 569

frozen in liquid N2, and ground into fine powder. 10 to 20 mg of the frozen powder from 570

each plant was mixed in the extraction solution by the ratio of 100µl extraction solution / 571

10mg frozen powder, containing 100mM MOPS, pH 7.0, 1mM EDTA, 5mM DTT, 572

glycerol, and protease inhibitors (Complete EDTA-free from Roche) with 0.5% Triton-X, 573

10% (v/v). After 5 minutes at 16,000g centrifugation, soluble protein extracts were 574



22

isolated, and which concentrations were quantified by using BCA protein assay kit 575

(Sigma). Equal abundance of soluble protein extract from each sample of each line was 576

pooled, and used for immunoblotting and Zymogram. 20 µg pooled soluble protein 577

extracts from each line were mixed with an equal volume of SDS loading solution (50 578

mM Tris, pH 6,8, 2% SDS, 0,1% bromophenol blue, 10% glycerol, and 5 mM DTT), 579

boiled 5 minutes, and loaded to 10 % SDS PAGEs. After electrophoresis at 10V cm-1 for 580

1-h at room temperature, proteins were transferred to PVDF membranes, which were 581

probed with monoclonal antibodies against YFP (Clontec JL-08) or SS4. Antibodies 582

against beta-actin (from Sigma) were added for quantitative internal controls. Two-color 583

IRDye® secondary antibodies and Odyssey®

Infrared Imaging System (Odyssey® CLx) 584

were then used to visualize signals for transformed proteins (Green) and beta-actin (Red). 585

Abundances of detected proteins were quantified by using Image Studio™ Lite software. 586

The remained homogenate was centrifuged for 5 min at 16,000 g at 4°C. Soluble proteins 587

in the supernatant were quantified using Bio-Red Protein Assay. For zymograms, 50 µg 588

protein extracts from each line were loaded onto 7.5% native PAGE gels containing 0.3% 589

oyster glycogen, following the procedures described in Crumpton-Taylor et al., 2013. 590

Starch Measurement 591

Whole rosettes of 25-day old plants from each line were harvested at the end of day and at 592

the end of night, decolorized in hot 70% (v/v) ethanol, stained with Lugol’s solution 593

(Sigma-Aldrich), and stored in cold water overnight to remove excess iodine from 594

rosettes. 595

To quantify starch content, whole rosettes of 25-day old plants of Col, ss4, and each of the 596

transformed lines was harvested at the end of day and at the end of night. Plants were 597

weighed, frozen in liquid N2, ground to a powder, and further homogenized in 2 ml of 598

1.12 M perchloric acid (HClO4). The homogenates were subjected to centrifugation 599

(3,000 g, 10 min, 20°C) to separate the insoluble and soluble fractions. The insoluble 600

fraction contained starch, and was washed once with cold water, four times with 70% 601

(v/v) ethanol, and resuspended in 1 ml water. Starch was quantified in the insoluble 602

fraction as described in Hostettler et al. (2011). 603



23

Confocal Microscopy 604

A 1 cm2 leaf piece from a 30 day-old plant was harvested at the middle of day and 605

observed by Zeiss LSM 780 confocal laser scanning with a 40x water immersion 606

objective. YFP-fused proteins were excited by a 488/514 nm argon laser, and detected at 607

520-570 nm. Chlorophyll auto-fluorescence was excited by a 594 nm helium neon laser, 608

and detected at 620-700 nm. 609

Isolation and Structural Analysis of Starch Granules 610

Whole rosettes of 5 week-old plants from Col, ss4, and each of the transformed lines were 611

harvested after an 11 h photoperiod, frozen in liquid N2, ground to a powder, 612

homogenized in starch extraction medium (50 mM Tris-HCl [pH 8], 0.2 mM EDTA and 613

0.5% [v/v] Triton X-100), and filtered through a series of nylon nets (100-µm, 30-µm, and 614

15-µm pore sizes). Starch granules were separated from the filtrate by centrifugation 615

(3,000 g, 20 min, at 20°C) over a 95% (v/v) Percoll (Sigma-Aldrich) cushion. Starch 616

pellets were washed three times with 0.5% (w/v) SDS and four times with water to 617

remove the remaining SDS. The pellets were dried for 16 h in vacuo. Half a milligram of 618

pure starch granules were boiled for 20 min, cooled at room temperature, debranched for 619

3 h by incubation in a solution containing 10 mM sodium acetate pH 4.8, 600 U 620

isoamylase (from Pseudomonas sp.; Sigma), and 1 U pullulanase (M1, from Klebsiella 621

planticola, Megazyme). Linear chains were analyzed by high performance anionexchange 622

chromatography with pulsed amperometric detection (HPAEC-PAD) as described in 623

Streb et al. (2008). 624

Light Microscopy and Electron Microscopy 625

Leaf pieces (2 mm2) were cut from three young rosette leaves from three 30-day old plants 626

and fixed for 16 h in 2% (v/v) glutaraldehyde and 0.05 M sodium cacodylate, pH 627

7.4, at 4°C. The leaf pieces were washed three times with 0.1 M sodium cacodylate, pH 628

7.4, and incubated overnight in 1% (w/v) osmium tetroxide with 0.1 M sodium 629

cacodylate, pH 7.4 at 4°C. Sequentially, pieces were washed three times with ice-cold 0.1 630

M sodium cacodylate, pH 7.4, and once with water. Samples were dehydrated in a series 631



24

of aqueous ethanol solutions from 50% (v/v) to 100% ethanol and once with 100% 632

acetone. The leaf pieces were incubated sequentially for 2 h in 25% (v/v) and 50% epoxy 633

resin (Spurr’s; Agar Scientific, in acetone), 16 h in 75% epoxy resin, and 7 h in 100% 634

epoxy resin. Embedding was completed by incubation for 48 h with fresh 100% epoxy 635

resin at 70°C. Semithin sections were cut with a Diatome 45° diamond knife. Five 636

hundred nm thick sections were placed on glass slides, stained for 10 min with toluidine 637

blue, washed with water and dried. Light micrographs were taken using Zeiss Axio 638

Imager Z2 light microscope with a 100x oil immersion objective. For transmission 639

electron microscopy, sections of 70-nm thickness were placed on formvar carbon-coated 640

copper grids, stained with 2% (w/v) uranyl acetate and Reynold’s lead citrate and imaged 641

with a FEI Morgagni 268 transmission electron microscope (TEM). Pictures are 642

representative for the analysis of sections from two individual plants per genotype. For 643

scanning electron microscopy (SEM), pure starch granules were coated with a 3 nm 644

platinum layer using Balzers MED010 coating device and visualized by Leo 1530 Gemini 645

microscope (Zeiss). 646

647

ACCESSION NUMBERS 648

Arabidopsis Genome Initiative gene codes for Arabidopsis STARCH SYNTHASE 4 (SS4) 649

is At4g18240. A. tumefaciens genome annotation for glycogen synthase (GS) is Atu4075. 650

651

SUPPLEMENTARY MATERIAL 652

Figure S1. The rosette leaf chosen for light and transmission electron microscopy. 653

Figure S2. Light micrographs of leaf sections of three independent lines transformed with 654

each construct encoding GS- or SS4-derived proteins. 655

Figure S3. Transmission electron micrographs of mesophyll cell chloroplasts of three 656

independent lines transformed with each construct encoding GS- or SS4-derived proteins. 657



25

Figure S4. Scanning electron micrographs of starch granules from three independent lines 658

transformed with each construct encoding GS- or SS4-derived proteins. 659

Figure S5. Transmission electron micrographs showing supernumerary granules in 660

chloroplasts of YFP-SS4N-GS lines. 661

Figure S6. Chain length distribution of starch from the wild type (Col), from ss4, and from 662

ss4 plants transformed with each construct encoding GS- or SS4-derived proteins. 663

Table S1. Distribution of starch granules in chloroplast sections of the wild type (Col), of 664

ss4, and of ss4 plants lines transformed with each construct encoding GS- or SS4-derived 665

proteins. 666

Table S2. Primers sequences used for ss4 mutant genotyping and for the amplification of 667

coding sequences of A. tumefaciens GS and A. thaliana SS4. 668

ACKNOWLEDGEMENTS 669

We thank Ángel Mérida for seeds of ss4-1 and for great suggestions during the course of 670

this work. We thank the staff at ScopeM (ETH Zurich) for advice on electron microscopy. 671

We thank Andrea Ruckle for assistance with plant cultivation. We thank Michaela 672

Fischer-Stettler for technical assistance with operation of HPAEC-PAD Dionex. We 673

thank David Seung for reading and providing insightful suggestions during manuscript 674

preparation. 675

676

FIGURE LEGENDS 677

Figure 1. Constructs used for the expression of GS- and SS4-derived proteins in ss4 678

mutants. Constructs encoding the full-length A. tumefaciens glycogen synthase (GS), the 679

C-terminal region of Arabidopsis SS4 (SS4C), and the N-terminal region of Arabidopsis 680

SS4 fused to GS (SS4N-GS) were cloned into vector pB7GWY2,0, modified to encode 681

the Arabidopsis Rubisco small subunit B chloroplast transit peptide (C in grey; amino 682

acids 1-53) upstream of YFP. Constructs encoding the N-terminal region of SS4 alone 683



26

(SS4N), the N-terminal region linked to GS (SS4N-GS), and the full length SS4 all 684

contained the endogenous SS4 chloroplast transit peptide (C in green; amino acids 1-42) 685

and were cloned into vector pB7WGY2,0, such that YFP was fused to their C-terminus. 686

Constructs were driven by the CaMV35s promoter (35S). Pnos:Bastar encoded a Basta 687

resistance gene, driven by the A. tumefaciens nopaline synthase gene promoter. Blue bars 688

and green bars indicate GS and SS4 regions, respectively. Both GS and the SS4C contain 689

glycosyltransferase 5 (GT5) and glycosyltransferase 1 (GT1) domains. The N-terminal 690

region of SS4 contains coiled-coil motifs. Protein domains were predicted by SMART 691

(Letunic et al., 2014). Numbers indicated amino acid residues of the corresponding 692

original full-length SS4 or GS protein sequences. 693

Figure 2. Detection of GS- and SS4-derived proteins in transformed ss4 plants. A, 694

Soluble proteins were extracted from Arabidopsis ss4 mutant plants transformed with the 695

constructs given in Figure 1. Equal amounts of protein from 4 plants of each line were 696

pooled, separated by SDS-PAGE and analyzed by immunoblotting with antibodies against 697

YFP (top panels) or SS4 (bottom panels). The expected molecular weights of the mature 698

introduced protein were: 80 kDa for YFP-GS, 94 kDa for YFP-SS4C, 85 kDa for SS4N-699

YFP, 137 kDa for SS4N-GS-YFP and YFP-SS4N-GS, and 141 kDa for SS4-YFP. In 700

some blots exposed for longer times, an endogenous 130-kDa protein in the wild type and 701

in ss4 mutants cross reacted with the anti-YFP antibody. The amounts of SS4-YFP and 702

endogenous SS4 are given as SS4/actin ratio below the anti-SS4 blot. B, For zymograms, 703

equal amounts of soluble protein were separated on native-PAGE gels containing 0.3% 704

(w/v) oyster glycogen. After electrophoresis, gels were incubated for 16 h in a solution 705

containing 1 mM ADP-glucose. Dark bands corresponding to the endogenous starch 706

synthase activities SS1 and SS3 were visible in all lines. Additional dark bands represent 707

the introduced synthase activities. Asterisks mark areas where very faint additional 708

synthase activities could be detected. 709

Figure 3. Growth and starch staining phenotype of ss4 plants expressing GS- or SS4-710

derived proteins. Plants grown in soil for 25 days under a 12-h day/12-h night cycle were 711

photographed at the end of the day (EOD). For iodine staining, rosettes harvested at the 712



27

EOD and end of night (EON) were decolourized with ethanol and stained with Lugol’s 713

solution. Col: wild-type plant. ss4: ss4-1 mutant plant. Plants designated _1, _2, and _3 714

are T2 individuals from independent transgenic lines, as indicated. The mean (± SE) 715

rosette fresh weight (Fw) for each line is given (n = 8-12; ND, not determined) and 716

designated as “a” or “b” when not significantly different from the wild type or ss4 mutant, 717

respectively (determined by ANOVA; p value < 0.05). 718

Figure 4. Starch contents of ss4 plants expressing GS- or SS4-derived proteins. Plants 719

grown for four weeks under 12-h day/12-h night cycles were harvested at the end of day 720

(white bars) and at the end of night (black bars). The mean starch content (± SE) for each 721

line is given (n = 4 individual rosettes for Col, ss4, and SS4N-YFP, n = 6 for all other 722

lines). For Col and for ss4, the data in the upper and lower panels are the same. The 723

percentage of starch broken down during the night is indicated. 724

Figure 5. The N-terminal region of SS4 confers the discoid shape of starch granules. Left 725

panels show light micrographs (LM) of toluidine blue-stained, semi-thin sections cut from 726

fixed, embedded tissue from leaf number 7 of each line. Middle panels show transmission 727

electron micrographs (TEM) of ultrathin sections cut from the same samples. Right panels 728

show scanning electron micrographs (SEM) of starch granules extracted from whole 729

rosettes of the given lines. The lines presented here are those showing the highest 730

expression of the introduced construct, except for YFP-GS, where the line with lowest 731

expression was chosen due to its relatively strong expression. Data from the remaining 732

lines is presented in Supplemental Figures S2, S3 and S4. White bars = 10 µm; black bars 733

= 2 μm. Black arrow, lenticular starch granule; red arrow, spherical starch granule. 734

Figure 6. Variation in granule morphology in chloroplasts of YFP-GS lines. Chloroplast 735

sections of expanding source leaves from each YFP-GS line were analyzed by LM and 736

TEM. A, LMs showing multiple granules in chloroplast sections. B, LMs showing zero, 737

one or two granules in chloroplast sections. C, LMs showing supernumerary granules in 738

chloroplast sections. D, TEM of chloroplasts with supernumerary granules. White bars = 739

10 µm; black bars = 2 µm. White arrows indicate supernumerary, irregular granules. 740



28

Figure 7. Variation in starch granule morphology in chloroplasts of SS4N-GS-YFP-741

expressing plants. LMs (left-hand panels) and TEMs (central and right-hand panels) of 742

chloroplast sections from expanding source leaves of the wild type (Col) and each of the 743

SS4N-GS-YFP-expressing lines. White bars = 10 µm; black bars = 2 µm. Black arrows, 744

lenticular type granules; white arrows: supernumerary granules; green arrows, smooth 745

supernumerary granules; red arrows, irregular supernumerary granules. 746

Figure 8. Sub-chloroplastic localization of YFP-tagged SS4 or GS proteins in mesophyll 747

cell chloroplasts from mature Arabidopsis leaves. Confocal fluorescence micrographs 748

showing free YFP and the YFP-tagged proteins (as indicated) are presented in the left-749

hand panels. The corresponding chlorophyll fluorescence is given in the central panels 750

and the merged images in the right-hand panels. Bars = 5 µm. 751

752

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Figure 1. Constructs used for the expression of GS- and SS4-derived proteins in ss4 mutants.Constructs encoding the full-length A. tumefaciens glycogen synthase (GS), the C-terminalregion of Arabidopsis SS4 (SS4C), and the N-terminal region of Arabidopsis SS4 fused to GS(SS4N-GS) were cloned into vector pB7GWY2,0, modified to encode the Arabidopsis Rubiscosmall subunit B chloroplast transit peptide (C in grey; amino acids 1-53) upstream of YFP.Constructs encoding the N-terminal region of SS4 alone (SS4N), the N-terminal region linked toGS (SS4N-GS), and the full length SS4 all contained the endogenous SS4 chloroplast transitpeptide (C in green; amino acids 1-42) and were cloned into vector pB7WGY2,0, such that YFPwas fused to their C-terminus. Constructs were driven by the CaMV35s promoter (35S).Pnos:Bastar encoded a Basta resistance gene, driven by the A. tumefaciens nopaline synthasegene promoter. Blue bars and green bars indicate GS and SS4 regions, respectively. Both GS andthe SS4C contain glycosyltransferase 5 (GT5) and glycosyltransferase 1 (GT1) domains. The N-terminal region of SS4 contains coiled-coil motifs. Protein domains were predicted by SMART(Letunic et al., 2014). Numbers indicated amino acid residues of the corresponding original full-length SS4 or GS protein sequences.

YFP‐GS

YFP‐SS4C

SS4N‐YFP

SS4N‐GS‐YFP

YFP‐SS4N‐GS

SS4‐YFP

YFP



Figure 2. Detection of GS- and SS4-derived proteins in transformed ss4 plants. A, Solubleproteins were extracted from Arabidopsis ss4 mutant plants transformed with the constructsgiven in Figure 1. Equal amounts of protein from 4 plants of each line were pooled, separated bySDS-PAGE and analyzed by immunoblotting with antibodies against YFP (top panels) or SS4(bottom panels). The expected molecular weights of the mature introduced protein were: 80 kDafor YFP-GS, 94 kDa for YFP-SS4C, 85 kDa for SS4N-YFP, 137 kDa for SS4N-GS-YFP andYFP-SS4N-GS, and 141 kDa for SS4-YFP. In some blots exposed for longer times, anendogenous 130-kDa protein in the wild type and in ss4 mutants cross reacted with the anti-YFPantibody. The amounts of SS4-YFP and endogenous SS4 are given as SS4/actin ratio below theanti-SS4 blot. B, For zymograms, equal amounts of soluble protein were separated on native-PAGE gels containing 0.3% (w/v) oyster glycogen. After electrophoresis, gels were incubatedfor 16 h in a solution containing 1 mM ADP-glucose. Dark bands corresponding to theendogenous starch synthase activities SS1 and SS3 were visible in all lines. Additional darkbands represent the introduced synthase activities. Asterisks mark areas where very faintadditional synthase activities could be detected.

B. SS4N‐GS‐YFP/ss4

SS1

SS3

YFP‐GS

YFP‐SS4N‐GS/ss4SS4N‐YFP/ss4 SS4‐YFP/ss4

*

SS4‐YFP/ss4

Col ss4 _1 _2 _3250

130

100

70

anti‐actin

anti‐SS4

SS4/actin

55

350.14 0.00 0.09 0.04 0.03

A.

Molecular weight

(kDa)

Molecular weight

(kDa)

*

ss4 _1

YFP‐GS/ss4

Col

250

130

100

70

_2 _3

YFP‐SS4C/ss4

Rubisco

_1 _2 _3

anti‐YFP SS4N‐YFP/ss4

ss4Col _1 _2 _3

SS4‐YFP/ss4

_1 _2 _3

SS4N‐GS‐YFP/ss4

_1 _2 _3ss4Col

YFP‐SS4N‐GS/ss4

_1 _2 _3ss4Col

*

Col ss4 _1 _2 _3 _1 _2 _3

YFP‐GS/ss4 YFP‐SS4C/ss4

_1 _2 _3 _1 _2 _3 _1 _2 _3 _1 _2 _3



Figure 3. Growth and starch staining phenotype of ss4 plants expressing GS- or SS4-derivedproteins. Plants grown in soil for 25 days under a 12-h day/12-h night cycle were photographedat the end of the day (EOD). For iodine staining, rosettes harvested at the EOD and end of night(EON) were decolourized with ethanol and stained with Lugol’s solution. Col: wild-type plant.ss4: ss4-1 mutant plant. Plants designated _1, _2, and _3 are T2 individuals from independenttransgenic lines, as indicated. The mean (± SE) rosette fresh weight (Fw) for each line is given(n = 8-12; ND, not determined) and designated as “a” or “b” when not significantly differentfrom the wild type or ss4 mutant, respectively (determined by ANOVA; p value < 0.05).

0.15 ± 0.01 ga

0.05 ± 0.00 gb

0.06 ± 0.00 gb

0.10 ± 0.01 g 0.06 ± 0.00 gb

0.06 ± 0.00 gb

0.10 ± 0.01 g 0.09 ± 0.01 gND 0.05 ± 0.00 gb

0.05 ± 0.00 gb

0.04 ± 0.00 gb

Fw

EOD

EON

Growth

Fw

EOD

EON

Growth

0.16 ± 0.01 ga

0.12 ± 0.01 g 0.12 ± 0.00 g 0.13 ± 0.01 ga

0.19 ± 0.01 g 0.20 ± 0.01 g0.09 ± 0.00 g 0.09 ± 0.00 g 0.11 ± 0.01 g

Col ss4 YFPYFP‐GS YFP‐SS4C SS4N‐YFP

_1 _2 _3 _1 _2 _3 _1 _2 _3

SS4N‐GS‐YFP SS4‐YFP

_1 _2 _3 _1 _2 _3

YFP‐SS4N‐GS

_1 _2 _3



Figure 4. Starch contents of ss4 plants expressing GS- or SS4-derived proteins. Plants grown forfour weeks under 12-h day/12-h night cycles were harvested at the end of day (white bars) and atthe end of night (black bars). The mean starch content (± SE) for each line is given (n = 4individual rosettes for Col, ss4, and SS4N-YFP, n = 6 for all other lines). For Col and for ss4, thedata in the upper and lower panels are the same. The percentage of starch broken down duringthe night is indicated.

0

2

4

6

8

10

12

Col ss4 GS 10 GS 8 GS 11 SS4C 1 SS4C 4 SS4C 5 SS4N 2 SS4N 3 SS4N 4

Starch

content(m

g g‐1FW

)

93% 50% 76% 72% 88% 72% 68% 68%

YFP‐GS YFP‐SS4C

_1 _3_2Col ss4

60% 53% 51%

SS4N‐YFP

_1 _3_2 _1 _3_2

_1 _3_2 _1 _3_2 _1 _3_2

0

2

4

6

8

10

12

Starch

content(m

g g‐1FW

)

93% 50% 90% 92% 96% 88% 89% 85%

YFP‐SS4N‐GS SS4‐YFPSS4N‐GS‐YFPCol ss4

86% 82% 81%



Col

ss4

YFP‐GS _1

YFP‐SS4C_3

SS4N‐YFP _1

SS4N‐GS‐YFP_3

YFP‐SS4N‐GS _1

SS4‐YFP_1

LM TEM SEM



Figure 5. The N-terminal region of SS4 confers the discoid shape of starch granules. Left panelsshow light micrographs (LM) of toluidine blue-stained, semi-thin sections cut from fixed,embedded tissue from leaf number 7 of each line. Middle panels show transmission electronmicrographs (TEM) of ultrathin sections cut from the same samples. Right panels show scanningelectron micrographs (SEM) of starch granules extracted from whole rosettes of the given lines.The lines presented here are those showing the highest expression of the introduced construct,except for YFP-GS, where the line with lowest expression was chosen due to its relatively strongexpression. Data from the remaining lines is presented in Supplemental Figures S2, S3 and S4.White bars = 10 µm; black bars = 2 μm. Black arrow, lenticular starch granule; red arrow,spherical starch granule.



Figure 6. Variation in granule morphology in chloroplasts of YFP-GS lines. Chloroplast sectionsof expanding source leaves from each YFP-GS line were analyzed by LM and TEM. A, LMsshowing multiple granules in chloroplast sections. B, LMs showing zero, one or two granules inchloroplast sections. C, LMs showing supernumerary granules in chloroplast sections. D, TEMof chloroplasts with supernumerary granules. White bars = 10 µm; black bars = 2 µm. Whitearrows indicate supernumerary, irregular granules.

YFP‐GS_1

YFP‐GS_2

YFP‐GS_3

A B C D



Figure 7. Variation in starch granule morphology in chloroplasts of SS4N-GS-YFP-expressingplants. LMs and TEMs of chloroplast sections from young leaves of the wild type (Col) and eachof the SS4N-GS-YFP-expressing lines. White bars = 10 µm; black bars = 2µm. Black arrows,lenticular type granules; white arrows: supernumerary granules; green arrows, smoothsupernumerary granules; red arrows, irregular supernumerary granules.

SS4N‐GS‐YFP_1

Col

SS4N‐GS‐YFP_2

SS4N‐GS‐YFP_3

E



YFP-SS4C_3

YFP

SS4N-YFP_1

Chlorophyll Merge

SS4N-GS-YFP_3

YFP-SS4N-GS_1

SS4-YFP_1

YFP-GS_1



Figure 8. Sub-chloroplastic localization of YFP-tagged SS4 or GS proteins in mesophyll cell

chloroplasts from mature Arabidopsis leaves. Confocal fluorescence micrographs showing free

YFP and the YFP-tagged proteins (as indicated) are presented in the left-hand panels. The

corresponding chlorophyll fluorescence is given in the central panels and the merged images in

the right-hand panels. Bars = 5 µm.



Parsed CitationsBall, S., Guan, H.P., James, M., Myers, A., Keeling, P., Mouille, G., Buléon, A., Colonna, P., and Preiss, J. (1996). From glycogen toamylopectin: a model for the biogenesis of the plant starch granule. Cell 86: 349–352.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ball, S., Colleoni, C., Cenci, U., Raj, J.N., and Tirtiaux, C. (2011). The evolution of glycogen and starch metabolism in eukaryotes givesmolecular clues to understand the establishment of plastid endosymbiosis. J. Exp. Bot. 62: 1775–1801.


Buléon, A., Colonna, P., Planchot, V., and Ball, S. (1998). Starch granules: structure and biosynthesis. Int. J. Biol. Macromol. 23: 85–112.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cuesta-Seijo, J.A., Nielsen, M.M., Ruzanski, C., Krucewicz, K., Beeren, S.R., Rydhal, M.G., Yoshimura, Y., Striebeck, A., Motawia, M.S.,Willats, W.G.T., and Palcic, M.M. (2016). In vitro biochemical characterization of all barley endosperm starch synthases. Front. Plant Sci.6: 1–17.


Crumpton-Taylor, M., Grandison, S., Png, K.M.Y., Bushby, A.J., and Smith, A.M.

(2012). Control of starch granule numbers in Arabidopsis chloroplasts. Plant Physiol. 158: 905–916.

Crumpton-Taylor, M., Pike, M., Lu, K.-J., Hylton, C.M., Feil, R., Eicke, S., Lunn, J.E., Zeeman, S.C., and Smith, A.M. (2013). Starchsynthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion. NewPhytol. 200: 1064–1075.


Delatte, T., Trevisan, M., Parker, M.L., and Zeeman, S.C. (2005). Arabidopsis mutants Atisa1 and Atisa2 have identical phenotypes andlack the same multimeric isoamylase, which influences the branch point distribution of amylopectin during starch synthesis. Plant J. 41:815–830.


Delvallé, D., Dumez, S., Wattebled, F., Roldán, I., Planchot, V., Berbezy, P., Colonna, P., Vyas, D., Chatterjee, M., Ball, S., Mérida, A., andD’Hulst, C. (2005). Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves. PlantJ. 43: 398–412.


Fontaine, T., Hulst, C.D., Maddelein, M.-L., Routier, F., Pepin, T.M., Decq, A., Wieruszeski, J., Delrue, B., Koornhuyse, N. Van Den,Bossu, J., Fournet, B., and Ball, S. (1993). Toward an understanding of the biogenesis of the starch granule. J. Biol. Chem. 268: 16223–16230.


Fujita, N., Yoshida, M., Kondo, T., Saito, K., Utsumi, Y., Tokunaga, T., Nishi, A., Satoh, H., Park, J.-H., Jane, J.-L., Miyao, A., Hirochika, H.,and Nakamura, Y. (2007). Characterization of SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIadeficiency in the rice endosperm. Plant Physiol. 144: 2009–2023.


Fujita, N., Yoshida, M., Asakura, N., Ohdan, T., Miyao, A., Hirochika, H., and Nakamura, Y. (2006). Function and characterization of starchsynthase I using mutants in rice. Plant Physiol. 140: 1070–1084.


Gámez-Arjona, F.M., Raynaud, S., Ragel, P., and Mérida, A. (2014). Starch synthase 4 is located in the thylakoid membrane and interactswith plastoglobule-associated proteins in Arabidopsis. Plant J. 80: 305–316.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Streb%2C S%2E%2C Delatte%2C T%2E%2C Umhang%2C M%2E%2C Eicke%2C S%2E%2C Schorderet%2C M%2E%2C Reinhardt%2C D%2E%2C and Zeeman%2C S%2EC%2E %282008%29%2E Starch granule biosynthesis in Arabidopsis is abolished by removal of all debranching enzymes but restored by the subsequent removal of an endoamylase%2E Plant Cell 20%3A 3448�??3466%2E&dopt=abstract

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http://scholar.google.com/scholar?as_q=Starch granule biosynthesis in Arabidopsis is abolished by removal of all debranching enzymes but restored by the subsequent removal of an endoamylase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Streb,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tetlow,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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Google Scholar: Author Only Title Only Author and Title

Zhang, X., Myers, A.M., and James, M.G. (2005). Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure andincrease the rate of starch synthesis. Plant Physiol. 138: 663–674.



http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang%2C X%2E%2C Myers%2C A%2EM%2E%2C and James%2C M%2EG%2E %282005%29%2E Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis%2E Plant Physiol%2E 138%3A 663�??674%2E&dopt=abstract

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Documents

1 STARCH SYNTHASE 4 Domain Functions 2 3 4€¦ · l., 2013; Ragel et al., 2013). These findings are . 100. consistent with a role for SS4 upstream of the other SSs, i.e., a role