2 In vitro - Plant Physiology€¦ · 32 Medicine, 480 Ray C. Hunt Dr., Charlottesville, VA 22908,...

Short Title 1

In vitro synthesis amp assembly of cellulose fibrils 2

3

4

Corresponding authors 5

B Tracy Nixon 6

Department of Biochemistry and Molecular Biology The Pennsylvania State University 7

University Park PA 16802 USA 8

Tel 814-863-4904 9

FAX 814-863-7024 10

e-mail btn1psuedu 11

12

Manish Kumar 13

Department of Chemical Engineering The Pennsylvania State University University Park PA 14

16802 USA 15

Tel 814-865-7519 16

FAX 17

e-mail manishkumarpsuedu 18

19

20

Journal Research Area 21

Biochemistry and Metabolism 22

23

Plant Physiology Preview Published on August 2 2017 as DOI101104pp1700619

Copyright 2017 by the American Society of Plant Biologists

wwwplantphysiolorgon August 15 2020 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

- 2 -

Synthesis and self-assembly of cellulose microfibrils from reconstituted cellulose synthase 24

25

Sung Hyun Cho1 Pallinti Purushotham

2 Chao Fang

3 Cassandra Maranas

4 Sara M Diacuteaz-26

Moreno5 Vincent Bulone

56 Jochen Zimmer

2 Manish Kumar

3 B Tracy Nixon

1 27

28

1Department of Biochemistry and Molecular Biology The Pennsylvania State University 29


2Department of Molecular Physiology and Biological Physics University of Virginia School of 31

Medicine 480 Ray C Hunt Dr Charlottesville VA 22908 USA 32

3Department of Chemical Engineering The Pennsylvania State University University Park PA 33

16802 USA 34

4Department of Chemical Engineering University of Washington Seattle WA 98105 35

5Division of Glycoscience School of Biotechnology Royal Institute of Technology (KTH) 36

Stockholm SE-10691 Sweden 37

6Australian Research Council Centre of Excellence in Plant Cell Walls School of Agriculture 38

Food and Wine University of Adelaide Waite Campus Urrbrae 5064 South Australia 39

Australia 40

41

One Sentence Summary 42

Liposome-reconstituted heterologously expressed cellulose synthases that contribute to 43

primarysecondary plant cell walls synthesized glucan chains that assembled into cellulose 44

microfibrils45


- 3 -

Footnotes 46

- Authorrsquos contributions 47

SHC MK and BTN designed the experiments interpreted the data and wrote the paper PP 48

and JZ constructed Pichia strains and performed radiolabeling assays SHC was involved in 49

most experiments SHC CF and CM conducted TEM analysis SMD and VB performed 50

linkage analysis 51

52

- Funding Information 53

This material is based upon work supported as part of The Center for LignoCellulose Structure 54

and Formation an Energy Frontier Research Center funded by the US Department of Energy 55

Office of Science and Office of Basic Energy Sciences under Award Number DE-SC0001090 56

57

Correspondence btn1psuedu manishkumarpsuedu 58

59


- 4 -

Abstract 60

Cellulose the major component of plant cell walls can be converted to bioethanol and is 61

thus highly studied In plants cellulose is produced by cellulose synthase a processive family-2 62

glycosyltransferase In plant cell walls individual β-14-glucan chains polymerized by CesA are 63

assembled into microfibrils that are frequently bundled into macrofibrils An in vitro system in 64

which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this 65

process Here we report the heterologous expression and partial purification of His-tagged 66

CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed 67

enrichment of PpCesA5 The recombinant protein was functional when reconstituted into 68

liposomes made from yeast total lipid extract The functional studies included incorporation of 69

radiolabeled Glc linkage analysis and imaging of cellulose microfibril formation using 70

transmission electron microscopy A number of microfibrils were observed either inside or on 71

the outer surface of proteoliposomes and strikingly several thinner fibrils formed ordered 72

bundles that either covered the surfaces of proteoliposomes or spawned from liposome surfaces 73

We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid 74

aspen These observations describe minimal systems of membrane-reconstituted CesAs that 75

polymerize β-14-glucan chains that coalesce to form microfibrils and higher-ordered 76

macrofibrils How these micro- and macrofibrils relate to those found in primary and secondary 77

plant cell walls is uncertain but their presence enables further study of the mechanisms that 78

govern the formation and assembly of fibrillar cellulosic structures and cell wall composites 79

during or after the polymerization process controlled by CesA proteins 80

81


- 5 -

Introduction 82

Cellulose is the most abundant biopolymer on earth (Pauly and Keegstra 2008 McNamara et 83

al 2015) It consists of bundles of β-14-glucan chains typically organized as microfibrillar 84

structures It is mostly found in plants bacteria oomycetes and green algae (Fugelstad et al 85

2009 John et al 2011 Augimeri et al 2015) Cellulose is the main structural component of 86

plant cell walls and is used for several technological applications including manufacturing of 87

paper textiles and furniture (McFarlane et al 2014) In recent years cellulose has become a 88

focus of those pursuing the production of sustainable biofuels due to its potential to be converted 89

to ethanol and other compounds (Pauly and Keegstra 2008) Detailed knowledge regarding the 90

structure and function of cellulose synthases (CesAs) and the mechanisms of cellulose 91

polymerization and microfibril assembly is likely to facilitate downstream processing of 92

cellulose (Cantarel et al 2009 McNamara et al 2015) 93

A simplified in vitro system that includes levels of CesA assembly sufficient to produce 94

cellulose microfibrils would greatly facilitate the acquisition of such knowledge There are many 95

CesA genes in plants whose products exhibit complex interactions (Popper et al 2011) For 96

example Arabidopsis thaliana has 10 CesA genes that are divided into two groups by the type of 97

cell wall that they make CesA1 -2 -3 -5 -6 and -9 are involved in primary cell wall synthesis 98

(Arioli et al 1998 Cano-Delgado et al 2003 Desprez et al 2007 Persson et al 2007) and 99

CesA4 -7 and -8 are involved in secondary cell wall synthesis (Taylor et al 2000 Brown et al 100

2005 Bosca et al 2006 Mendu et al 2011 McFarlane et al 2014) These CesAs form a 101

hexameric rosette structure in the plasma membrane called Cellulose Synthase Complex (CSC) 102

and it was believed until recently that each of the six lobes have six subunit CesA proteins 103

(Kimura et al 1999 Doblin et al 2002 Cosgrove 2005 Somerville 2006) The latest 104

developments based on imaging structural and computational approaches suggest that each CSC 105

lobe possesses three CesA molecules (Newman et al 2013 Hill et al 2014 Nixon et al 2016 106

Vandavasi et al 2016) A single CSC should thus produce up to 18 single glucan chains 107

presuming that all individual CesA proteins are active synthases these chains subsequently 108

crystallize to form cellulose microfibrils that are 3-5 nm in diameter (Ha et al 1998 Cosgrove 109

2005 Thomas et al 2013) Higher order bundles of microfibrils are also seen in cell walls 110

(Marga et al 2005 Somerville 2006 Zhang et al 2016) 111


- 6 -

While assembly of CesAs and assembly of glucan chains involve different macromolecules 112

they are likely to be linked processes but the extent of linkage remains unclear One scenario is 113

that the CesA enzymes assemble into trimeric lobes and these into rosette CSCs Subsequent 114

glucan synthesis and extrusion from the organized rosette provides chains to form fibrillar 115

cellulose Alternatively CesA might assemble into trimeric lobes that each synthesize three 116

adjacent glucan chains which self-assemble into a sub-microfibril The latter further assemble 117

into complete microfibrils providing feedback onto individual lobes moving them into a rosette 118

distribution The microfibrils could then further assemble with each other and wall matrix 119

materials 120

Some clues have been garnered regarding assembly of CesA proteins The secondary 121

structure of most plant CesA proteins consists of at least 6 transmembrane helices (TMHs) that 122

anchor and separate a cytoplasmic glycosyltransferase (GT) domain from an N-terminal 123

intrinsically unfolded domain (Sethaphong et al 2013) In addition to possessing the catalytic 124

function the GT domain includes a plant conserved region (P-CR) and a class specific region 125

(CSR) (Slabaugh et al 2014) The catalytic function is provided in part by three broadly 126

conserved aspartate-containing motifs designated as DDG DCD and TED followed by a 127

conserved QXXRW motif (Saxena and Brown Jr 1997 Saxena et al 2001 Sethaphong et al 128

2013) In vitro studies have shown that the catalytic domain of rice CesA1 forms redox-sensitive 129

dimers (Olek et al 2014) and that the same region of Arabidopsis thaliana CesA1 forms redox-130

insensitive trimers (Vandavasi et al 2016) Small-angle X-ray scattering (SAXS) and 131

transmission electron microscopy (TEM) based low-resolution volumes of the trimer fit well to 132

averaged lobe volumes from P patens rosettes imaged by freeze-fracture TEM (Nixon et al 133

2016) A Zn2+

-binding RING domain is encoded in the N-terminal regions of CesAs which may 134

be responsible for dimerization of CesA proteins (Kurek et al 2002) 135

Superimposed on this as yet poorly defined tendency for CesA subunits to assemble is an 136

intrinsic propensity for nascent β-14-glucan chains to form microfibrils with varied packing of 137

the chains (Ha et al 1998 Cosgrove 2005 Thomas et al 2013) Cellulose microfibrils are 138

deposited in transverse direction with respect to the cell growth (Szymanski and Cosgrove 2009) 139

and reoriented during cell wall expansion (Baskin 2005 Anderson et al 2010) Within a 140

microfibril the cellulose chains are held together by hydrogen bonds and Van-der-Waals forces 141

(Nishiyama et al 2002 Nishiyama et al 2003) In nature parallel chains are packed with low 142


- 7 -

order hence called lsquoamorphousrsquo or with high order as found in crystalline cellulose Because 143

individual β-glucan chains are synthesized as a flat ribbon with 180-degree flipping of the 144

orientation of successive glucose monomers it is possible to pack them in crystalline sheets with 145

the lateral register of the lsquouprsquo or lsquodownrsquo glucose moieties in adjacent chains in different ways 146

giving rise to the Iα and Iβ crystalline forms The Iα form is mostly found in bacterial and algal 147

cellulose and the Iβ form in higher plants and tunicate animals (Atalla and Vanderhart 1984 148

Belton et al 1989) A single microfibril may contain a mixture of these forms and interactions 149

between microfibrils contribute significantly to the mechanical properties of cellulose The 150

parallel alignment of glucan chains is consistent with simultaneous synthesis of cellulose chains 151

in CSCs (Somerville 2006) It is not known if CesA facilitates the close placement of cellulose 152

chains to enable hydrogen bonding and hydrophobic interactions if the close distance between 153

nascent glucan chains is sufficient to induce the formation of the higher order cellulose 154

microfibrils or if other protein(s) is(are) required for this assembly (Somerville 2006) 155

Recent work demonstrated that a single CesA isoform (CesA8 from hybrid aspen PttCesA8) 156

synthesizes cellulose in vitro and packs it into fibrils observable by TEM (Purushotham et al 157

2016) (Purushotham et al 2016) PttCesA8 contributes to the formation of secondary cell walls 158

Here we report similar synthesis of cellulose microfibrils by reconstituted CesA5 of 159

Physcomitrella patens (PpCesA5) which is involved in primary cell wall formation (Goss et al 160

2012) Previously we had shown that overexpression of PpCesA5 in P patens itself yielded 161

partially purified protein that synthesized cellulose microfibrils but the presence of other CesA 162

isoforms and accessory proteins could not be ruled out (Cho et al 2015) Now we eliminate the 163

possible presence of additional isoforms and other proteins by purifying heterologously 164

expressed PpCesA5 from Pichia pastoris and reconstituting it into proteoliposomes 165

Reconstituted PpCesA5 produced cellulose microfibrils demonstrated by incorporation of 166

radioactive Glc from UDP-[3H]-Glc cellulase specific degradation TEM analysis and linkage 167

analysis Furthermore TEM analysis identified cellulose microfibrils within and on the surface 168

of proteoliposomes bearing PpCesA5 or PttCesA8 These microfibrils coalesced into higher 169

order lsquomacrofibrilsrsquo These results confirm that single isoforms of CesAs for primary and 170

secondary cell wall synthesis are sufficient to produce cellulose microfibrils and suggest that 171

glucan chains can form higher order cellulose structures by self-assembly without the need for 172

accessory proteins 173


- 8 -

174

Results 175

176

Heterologous expression and purification of PpCesA5 177

PpCesA5 is predicted to have 7 transmembrane domains an N-terminal Zn-binding domain 178

and a long cytosolic region between the second and the third transmembrane helices 179

(Supplemental Fig S1) The cytosolic region contains P-CR and CSR regions as well as a TED 180

motif the latter believed to deprotonate the acceptor hydroxyl via its aspartic acid residue 181

(Morgan et al 2014) PpCesA5 conjugated with 12x His-tag at the C-terminus was 182

heterologously expressed in Pichia Expression in Pichia was directed by using methanol to 183

induce transcription from the alcohol oxidase 1 (AOX1) promoter Membrane proteins were 184

isolated and further purified with TALON (cobalt) resin PpCesA5 proteins were co-purified 185

with other membrane proteins as shown by immunoblot using anti-PpCesA5 antibody 1 (Lanes 186

1-3 Fig 1) The final elution fraction contained a highly enriched PpCesA5 protein of ~125 kDa 187

(Lane 9 Fig 1) Two other PpCesA5 specific antibodies 2 and 3 also detected similar band 188

patterns whereas anti-His antibody identified an additional band at around 55 kDa 189

(Supplemental Fig S2) While the epitopes of the PpCesA5 specific antibodies were in the 190

cytosolic domain (Supplemental Fig S1) the His-tag was at the C-terminus suggesting that the 191

N-terminal region is more prone to degradation Similarly heterologously expressed PttCesA8 192

also showed N-terminal degradation that was speculated to be due to conformational flexibility 193

or loose association of the N-terminal RING finger region (Purushotham et al 2016) 194

To further confirm that PpCesA5 was enriched proteins in the region between 110-140 kDa 195

were excised from an SDS-PAGE gel and analyzed by mass spectrometry (MS) Three peptides 196

of PpCesA5 including 444VQPTFVK450 538AGAMNALVR546 and 808GSAPINLSDR817 were 197

identified together with fragments of 18 proteins from Pichia including the catalytic subunit of 198

β-13-glucan synthase (Supplemental Table S1) 199

200

PpCesA5 is functional when reconstituted in proteoliposomes 201

Since CesA proteins are integral membrane proteins the enriched PpCesA5 was 202

reconstituted into proteoliposomes using total lipid extract from yeast These proteoliposomes 203

were then subjected to biochemical characterization using radiolabeled UDP-[3H]Glc as a tracer 204


- 9 -

followed by detergent disruption and descending paper chromatography (Fig 2) as described 205

(Omadjela et al 2013 Purushotham et al 2016) Radiolabeled glucose was incorporated into 206

insoluble material as shown by radioactivity that failed to leave the origin indicating that 207

PpCesA5s in proteoliposomes were functional 208

CesA proteins require cations as cofactors for function Activity of PpCesA5 209

proteoliposomes was tested with different cations including Ca2+

Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+

was most effective and that Zn2+

in combination with Mn2+

appeared to have no 211

synergistic effect (Fig 2A) These results were consistent with those from PttCesA8 212

(Purushotham et al 2016) Mn2+

alone was used in all subsequent tests 213

The incorporation of radiolabeled Glc from UDP-[3H]-Glc into synthesized product was 214

monitored over time (Fig 2B) The radioactive signal reached saturation at 150 min which 215

might be due to the depletion of substrate loss of protein activity or product inhibition In 216

contrast it took 90 min for PttCesA8 to reach a plateau (Purushotham et al 2016) Enzyme 217

kinetics assays were conducted using a constant level of UDP-[3H]-Glc with variation of UDP-218

Glc concentration These assays show monophasic Michaelis-Menten kinetics with KM = 137 219

(102 - 179) microM (Fig 2C 95 confidence interval in parenthesis) 220

To confirm that the in vitro product contained cellulose it was treated with β-14-glucanse 221

solubilizing 73 of the radiolabeled product (Fig 2D) The enriched preparation of PpCesA5 222

contained other proteins from Pichia that were present in the 110-140 kDa region of an SDS-223

PAGE gel (Supplemental Table S1) including the catalytic subunit of β-13-D-glucan synthase 224

(callose synthase) To examine potential contribution of callose synthase to the incorporated 225

radioactive product signal in vitro products were subjected to treatment with β-13-glucanase 226

solubilzing 27 of the radiolabeled product (Fig 2D) 227

To further confirm that the observed signal resulted from PpCesA5 activity we introduced 228

amino acid substitution D782N of the catalytically crucial TED motif of PpCesA5 (Supplemental 229

Fig S3) The analogous substitution inactivated PttCesA8 (Purushotham et al 2016) As evident 230

in immuno-blots PpCesA5(D782N) was sensitive to proteolysis Proteoliposomes prepared as 231

for the wildtype PpCesA5 showed no capacity to incorporate [3H]-Glc (Supplemental Fig S3) 232

233


- 10 -

In vitro-produced glucan chains assemble into cellulose microfibrils 234

β-14-D-glucan chains produced from CesAs form microfibrils and these are sometimes 235

bundled into larger arrays To determine if β-14-glucan chains produced by PpCesA5 236

proteoliposomes form microfibrils or higher order bundles proteoliposomes that had been 237

incubated with UDP-Glc were examined over time by TEM (Fig 3) Individual glucan chains are 238

too small to be seen with this method but microfibrils are large enough and they were first 239

observed within 5 min of incubation with UDP-Glc The initial amount of microfibrils increased 240

upon incubation for 60 and 180 min There were no microfibrils formed in a negative control 241

reaction lacking UDP-Glc nor any jagged-edge fibrils as reported for callose (Him et al 2001) 242

Repeated experiments with the TED mutant protein PpCesA5(D782N) yielded no observable 243

microfibrils (Supplemental Fig S5) Pre-treatment of proteoliposomes bearing wild-type 244

PpCesA5 with Triton X-100 caused them to fail to yield microfibrils (Supplemental Fig S6) 245

Also the addition of Zn2+

to PpCesA5-proteoliposomes had no effect on the level of microfibril 246

formation (Supplemental Fig S7) The thickness of microfibrils made by PpCesA5 was 247

estimated by cryo-TEM and found to be 43plusmn08 nm (Supplemental Fig S8) Most microfibrils 248

disappeared after 15 min of incubation with cellulase while a negative control without cellulase 249

showed no loss of microfibrils (Fig 4) 250

To further confirm that the fibrils were cellulose microfibrils with 14-glucose linkage a 251

glycosidic linkage analysis was performed by permethylation followed by gas chromatography 252

coupled to electron-impact mass spectrometry (GCEI-MS) In vitro products from a large scale 253

reaction were pretreated with α-amylase and amyloglucosidase to remove Pichia-derived 254

glycogen-like α-14-glucan chains Thereafter the sample was permethylated to alditol acetates 255

and then subjected to GCEI-MS analysis Comparison with reference peaks confirmed that the 256

sample mainly contained 14-D-glucose with no 13-D-glucose being observed (Fig 5 257

Supplemental Fig S9A) The sample also contained a small level of terminal glucose as further 258

identified by electron-impact mass spectrometry (Supplemental Fig S9B) Analogously treated 259

material that was produced by proteoliposomes bearing the inactive PpCesA5(D782N) showed 260

no 14-D-glucose (Supplemental Fig S10A) However 14-D-glucose was present prior to 261

eliminating α-14-glucans with amylase (Supplemental Fig S10B) 262

263


- 11 -

Higher order self-assembly of cellulose microfibrils 264

It is known that cellulose microfibrils are often bundled into larger fibrils in plant cell walls 265

To observe possible higher order assembly that might reflect a bundling process in the PpCesA5 266

and PttCesA8proteoliposome systems we thoroughly explored negatively stained TEM grids 267

with in vitro products to find unbroken or partially broken proteoliposomes This revealed 268

cellulose microfibrils near and apparently attached to the surface of proteoliposomes with 269

PpCesA5 as if they were spawned from the proteoliposomes (Fig 6A and Fig 6B) The 270

thicknesses of such fibrils varied with thinner fibrils merging to form thicker fibrils We also 271

found PpCesA5-proteoliposomes that contained numerous cellulose microfibrils inside and a few 272

microfibrils exiting out through the membranes which also formed higher order assemblies 273

outside (Fig 6C) Some cellulose microfibrils wrapped around the proteoliposomes (Fig 6D) In 274

many images thin cellulose fibrils coalesced into higher order micro- or macrofibrils (Fig 6 275

panels C E and F) which in some cases might be considered bundles This coalescence was also 276

observed in cellulose microfibrils produced by proteoliposomes bearing PttCesA8 (Fig 7) Such 277

lsquobundledrsquo microfibrils were also observed in other grid areas (Fig 4 panels A and G marked by 278

arrowheads) In total the area of lsquobundledrsquo microfibrils accounted for 36-43 of the 279

microfibrillar area in each image These lsquobundledrsquo microfibrils were also eliminated by cellulase 280

digestion prior to imaging (Fig 4H) indicating that they were cellulose microfibrils 281

282

Discussion 283

In this study proteoliposomes bearing single isoforms of CesAs known to synthesize primary 284

cell walls was able to synthesize β-14-glucan chains that were assembled into cellulose 285

microfibrils The cellulose microfibrils were often seen to merge into higher order forms It is not 286

yet clear if these in vitro products relate to the microfibrils macrofibrils or bundles seen in plant 287

cell walls so in the following discussion we will explicitly denote the in vitro cellulose 288

assemblies with superscript lsquoivrsquo (thus microfibrilsiv

macrofibrilsiv

or bundlesiv

) 289

Both biochemical and TEM data showed the synthesis of β-14-glucan chains that 290

assembled into microfibrilsiv

Incorporation of [3H]-Glc from UDP-[

3H]-Glc into insoluble 291

material that was sensitive to cellulase the presence of cellulose-like fibers with diameters of 43 292

nm in negative stain and cryo TEM images and the presence of β-14-D-Glc in linkage analysis 293

in the in vitro product confirm the synthesis of cellulose microfibrilsiv

In addition to the 294


- 12 -

synthesis of cellulose there was some evidence for the production of β-13-glucan chains The 295

presence of a 110-140 kDa fragment of the 220 kDa β-13-glucan synthase of Pichia and 296

observed partial digestion of in vitro synthesized insoluble polymers by β-13-glucanase are 297

consistent with callose synthesis However we observed no fibers in negative stain images that 298

had jagged edges as reported for callose and the linkage analysis showed no evidence of β-13-D-299

glucose These contradictory observations could be explained by variable presence of functional 300

Pichia β-13-glucan synthase in the PpCesA5 preparations of this study Such variation was seen 301

for preparations of PttCesA8 of hybrid aspen (Purushotham et al 2016) 302

A key aspect of our previous report on PttCesA8 and this report on PpCesA5 is that a single 303

isoform of plant CesA can alone produce β-14-glucan chains of cellulose These observations 304

force new considerations regarding the prior models of CSC composition We note that the CSC 305

is a complex of CesA proteins arranged in tightly bound lsquolobesrsquo that are more loosely contained 306

within mostly hexamers (sometimes pentamers) of lobes (Kimura et al 1999 Desprez et al 307

2007 Nixon et al 2016) Recent evidence strongly suggests that each lobe is a trimer of CesA 308

and until recently these trimers were assumed to be heterotrimers of different CesA isoforms the 309

exact isoforms depending on whether the CSC is involved in making cellulose for primary versus 310

secondary cell walls (Cosgrove 2005) The genetic redundancy has been interpreted as a way 311

plants provide differential regulation of cellulose synthesis in specific tissues and developmental 312

events (McFarlane et al 2014) The results presented here for CesA5 from the non-vascular 313

plant P patens and previously for CesA8 from the vascular plant hybrid aspen undeniably show 314

that a single isoform is capable of forming cellulose microfibrilsiv

in in-vitro systems This is 315

thus true for CesAs known for contributing in vivo to the production of primary (PpCesA5) or 316

secondary (PttCesA8) cell walls These observations of single isoforms making cellulose 317

microfibrilsiv

are consistent with reported 111 stoichiometric presence of CesA isoforms needed 318

for normal primary and secondary cell wall formation (Gonneau et al 2014 Hill et al 2014) 319

but they do raise the possibility of homomeric complexes 320

If one only considers the moss P patens it could be argued that homomeric lobes may be 321

restricted to non-vascular plants This would be reasonable because the P patensrsquo genome has 322

seven nearly identical CesA genes (Roberts and Bushoven 2007) and also has CSCs in the 323

plasma membranes similar to the rosettes seen in vascular plants (Roberts et al 2012 Nixon et 324

al 2016) Based at least in part on such observations it was predicted that a single CesA 325


- 13 -

isoform might be able to substitute for other CesAs in the CSCs of P patens Consistent with 326

that prediction single knock-out of PpCesA6 or PpCesA7 showed no phenotype only the double 327

knock-out resulted in reduction of stem lengths (Wise et al 2011) However our concurrent 328

study of hybrid aspen PttCesA8 that was also heterologously expressed in Pichia and 329

reconstituted into proteoliposomes makes it clear that only one isoform of CesA from vascular 330

plants is sufficient to yield cellulose microfibrilsiv

(Purushotham et al 2016) In both the moss 331

and poplar studies CesA in its detergent-solubilized form failed to incorporate Glc from UDP-332

Glc into glucan chains regaining this function only when incorporated into proteoliposomes The 333

amino acid sequences of CesA proteins in hybrid aspen are diverse like those in Arabidopsis 334

(Djerbi et al 2004) Thus we propose that CesA proteins will generally need to be in a lipid 335

bilayer to adopt functional conformation(s) and that they can assemble into homotrimer as well 336

as heterotrimer lobes (the latter not yet having been demonstrated in vitro) 337

In plants the individual β-14 glucan chains made by CesA proteins are assembled into 338

microfibrils and these into macrofibrils or higher order bundles such as those noted in atomic 339

force microscopic images of onion epidermal cell walls (Zhang et al 2016) The relationship 340

between trimeric lobes in rosettes and assembly of crystalline cellulose microfibrils is not yet 341

understood It is very intriguing that we observe micro- and macrofibrilsiv

in both the CesA 342

studies including the one reported here for CesA5 and in the CesA8 studies presented earlier 343

(Purushotham et al 2016) Such microfibrils are not made by the bacterial cellulose synthase 344

BcsA-BcsB even when present at high concentrations (Purushotham et al 2016) However 345

when BscA-BcsB was immobilized on a nickel-film followed by reaction with UDP-Glc 346

fibrillar cellulose was produced (Basu et al 2016 Basu et al 2017) This suggests that close 347

proximity of cellulose synthase proteins is required and sufficient for formation of microfibrilsiv

348

and we infer that such close proximity is present in the membrane-reconstituted moss and hybrid 349

aspen synthases CesA5 and CesA8 350

The lateral dimension of an individual cellulose glucan chain is lower than 05 nm which 351

makes isolated chains invisible in negative stain TEM images As shown in Fig 6 and Fig 7 352

variably thick fibers are seen and these tend to coalesce into larger fibrils While negative stain 353

images do not give reliable size estimates these relative differences in size are clearly present 354

We think it possible that a homotrimer of CesA5 or CesA8 is capable of making an elemental 355

fibril of three glucan chains and that these can further assemble to form more typical 356


- 14 -

microfibrils of about 43 to 48 nm in diameter The estimated diameter presented here from 357

cryo-TEM images is reliable given the absence of any staining and such sizes are consistent 358

with diameters reported for plant cell walls (Ha et al 1998 Cosgrove 2005 Thomas et al 2013 359

Zhang et al 2016) This raises the possibility that homotrimer lobes could be organized into 360

higher order rosettes as the microfibrilsiv

are formed It is simultaneously possible that the 361

dimerization propensity of N-terminal domains of CesA could help organize rosette formation by 362

bringing together lobes (Kurek et al 2002) Indeed truncation of N-terminal Zn2+

-binding 363

domains from PttCesA8 resulted in the formation of few or no microfibrils despite significant 364

production of β-14-glucan chains (Purushotham et al 2016) Future use of these in vitro 365

systems may reveal the membrane distributions of wild-type and mutant CesAs before during 366

and after catalysis with UDP-Glc and thus allow testing these hypotheses 367

368

Conclusions 369

PpCesA5 of the moss P patens was successfully expressed heterologously in Pichia and 370

partially purified followed by reconstitution into proteoliposomes Proteoliposomes bearing 371

PpCesA5 synthesize glucan chains that assembled into higher order cellulose microfibrilsiv

and 372

macrofibrilsiv

or bundlesiv

comparable to what is seen for a vascular plant CesA We discuss how 373

formation of cellulose microfibrils from single isoforms of CesA could be attributable to close 374

proximity of CesA proteins in lipid bilayers formation of higher order complexes among CesAs 375

or a combination of that and feedback from glucan chain crystallization ndash these assembly 376

processes remain to be investigated These systems for reconstitution of functional cellulose 377

synthases in vitro serve as foundations for further studies on the synthesis of cellulose by CesA 378

proteins and the assembly of nascent glucan chains into elementary fibrils microfibrils and 379

microfibril bundles in the absence or presence of matrix polysaccharides Access to purified 380

membrane-embedded functional CesAs may also yield structures of CesA from non-vascular 381

and vascular plants 382

383

Materials and Methods 384

385

Cloning and transformation of PpCesA5 386


- 15 -

The PpCesA5 gene was amplified by PCR using a primer set designed to add 12 x His tag at 387

the C-terminus 5rsquo-ACTAATCAAGGTACCATGGAGGCTAATGCAGGCCTTATT-3rsquo 5rsquo- 388

ACAATAGCGGCCGCTCAGTGATGGTGATGGTGATGGTGATGGTGATGGTGATGACA389

GCTAAGCCCGCACTCGAC -3rsquo Once synthesized the PCR product was digested with KpnI 390

and NotI restriction enzymes The resulting fragment was ligated into KpnI and NotI-linearized 391

pPICZ A vector Five microg of pPICZ A plasmid containing PpCesA5 was used to transform into 392

the P pastoris strain SMD1168H For preparation of the SMD1168H cells for transformation a 393

colony grown on YPDS plate containing 1 yeast extract 2 peptone and 2 glucose was 394

inoculated into 50 mL YPDS liquid medium followed by culture at 30degC by shaking until 395

growth reached early stationary phase (~06-2 x 108

cellsmL) Cells were harvested by 396

centrifugation at 3000 rpm for 5 min at 4degC followed by washing with 40 mL ice-cold sterile 397

water and centrifugation at 2500 rpm for 5 min at 4degC After washing with 20 mL ice-cold water 398

cells were resuspended in 5 mL ice-cold sorbitol solution (2 sorbitol in water) and pelleted at 399

2000 rpm for 5 min at 4degC The cells were resuspended in 150 microL ice-cold sorbitol solution from 400

which 40 microL of cells were mixed with 5 microg PmeI-linearized DNA in a pre-chilled electroporation 401

cuvette Electroporation was performed at 15 kV 25 microF and 200 Ohms for 5 sec which was 402

immediately followed by addition of 1 mL 1 M ice-cold sorbitol solution Transformed cells 403

were screened on YPDS medium containing 100 microgml of Zeocin 404

The catalytically inactive PpCesA5 construct was generated using the QuikChange 405

mutagenesis kit (ThermoFisher Scientific) following the manufacturerrsquos protocol Primers used 406

for mutagenesis are 5rsquo-CTGTCACCGAGAATATTCTGACGG-3rsquo and 5rsquo-407

CCGTCAGAATATTCTCGGT GACAG-3rsquo 408

409

Enrichment of PpCesA5 and PttCesA8 410

PpCesA5 and PttCesA8 were partially purified and reconstituted to proteoliposomes 411

following the method previously described (Purushotham et al 2016) The Pichia pastoris strain 412

expressing PpCesA5 or PttCesA8 was grown on YPDS medium at 30degC overnight A colony 413

was inoculated into a 500 mL pre-culture medium containing 1 yeast extract 2 peptone 1 414

glycerol 034 yeast nitrogen base 1 ammonium sulfate and 000004 biotin followed by 415

shaking incubation at 30degC overnight Cells were collected by centrifugation at 2600 x g at 4degC 416

for 15 min and resuspended in 1 L of induction medium (1 yeast extract 2 peptone 07 417


- 16 -

methanol 034 yeast nitrogen base 1 ammonium sulfate and 000004 biotin) to an OD600 418

of 05 followed by shaking incubation at 30degC for 24 h Grown cells were collected by 419

centrifugation and frozen at -80degC until use Frozen cells were thawed in a Cell Resuspension 420

Buffer containing 20 mM Tris-HCl pH 75 1 M sorbitol 1x EDTA-free protease inhibitor tablet 421

(Thermo Scientific) and 1 mM phenylmethylsulfonyl fluoride (PMSF) and lysed by three passes 422

using a microfluidizer at 20000 psi The lysate was centrifuged at 10000 g at 4degC for 10 min 423

and its supernatant was subjected to ultracentrifugation at 200000 x g at 4degC for 2 h The pellet 424

corresponding to vesicle fraction was resuspended in 50 mL Membrane Resuspension Buffer 425

containing 150 mM sodium phosphate pH 75 100 mM sodium chloride 40 mM n-dodecyl-β-D-426

maltoside (DDM) 10 glycerol 1x EDTA-free Protease inhibitor tablet (Thermo Scientific) and 427

1 mM PMSF followed by gentle agitation at 4degC for 1 h Insoluble materials removed by 428

ultracentrifugation at 200000 x g at 4degC for 40 min The obtained membrane protein fraction 429

was incubated with 5 mL pre-equilibrated TALON Superflow Resin (GE Healthcare) with gentle 430

agitation at 4degC overnight The mix was applied to an empty column and washed with in a series 431

of 15 mL washing buffer (150 mM sodium phosphate pH 75 100 mM sodium chloride and 1 432

mM LysoFos Choline Ether 14) containing 20 40 60 or 80 mM imidazole Protein was eluted 433

by 15 mL washing buffer containing 350 mM imidazole Imidiazole was reduced to less than 05 434

mM by repeated concentrationdilution cycles through a Vivaspin 20 desalting column (30 kDa 435

cutoff GE Healthcare) All fractions were examined by western blot analysis 436

437

Reconstitution of proteoliposomes 438

Chloroform was evaporated from a pre-dissolved yeast total lipid extracts (Avanti Polar 439

Lipids) by a stream of nitrogen gas which was then completely dried by overnight incubation in 440

a vacuum chamber The lipid (100 mg) was resuspended in 1 mL of 120 mM 441

lauryldimethylamine oxide (LDAO) solubilized by heating at 60degC for 1 h and stored at -20degC 442

until use Bio-Beads SM-2 Adsorbent Media (Bio-Rad) was washed in deionized water at room 443

temperature for 10 min and dried on a filter paper Six hundred microL of partially purified PpCesA5 444

or PttCesA8 was mixed with 400 microL yeast lipid to which dried Bio-Beads SM-2 Adsorbent 445

Media was added to 75 of the total volume This mix was incubated overnight with gentle 446

rotation at 4degC Reconstitution was determined by visual turbidity 447

448


- 17 -

Immunoblot analysis 449

Protein fractions were separated on a 4-12 gradient PAGE gel (GenScript) and transferred 450

to a nitrocellulose membrane (GE Healthcare) which was hybridized with one of three 451

monoclonal PpCesA5-specific antibodies raised against the synthetic peptides 452

681CKFSRKKTAPTRSDS694 506CGHSGGHDTDGNELP519 or 473CAQKVPEEGWTMQDG486 453

(GenScript) The hybridized blot was incubated with secondary antibody conjugated with 454

horseradish peroxidase (HRP) Chemiluminescent signals generated by Super Signal West Dura 455

Extended Duration Substrate (Thermos Scientific) were detected by a GelLogic 4000 Pro System 456

(Carestream Health) 457

458

Activity assay 459

In general reconstituted proteoliposome (PL) was mixed with a reaction buffer containing 20 460

mM Tris-HCl pH75 100 mM sodium chloride 10 glycerol 20 mM manganese chloride 3 461

mM UDP-Glc and 025 μCi UDP-[3H]-Glc followed by incubation at 37degC for 3 h However 462

assays with alternate components and conditions were performed as indicated in each 463

experiment Reactions were stopped by adding triton X-100 to a final concentration of 01 The 464

reaction mix was then centrifuged at 15000 rpm for 20 min to collect the product which was 465

resuspended in 20 μL of cellulose resuspension buffer containing 20 mM Tris-HCl pH75 100 466

mM sodium chloride and 10 glycerol and applied to a descending chromatography paper 467

(Whatman 2MM) using 60 ethanol Following air-dry the chromatography paper was 468

submerged in 5 mL ScintiVerse BD Cocktail (Fisher Scientific) and radioactivity was measured 469

in a Beckman Coulter LS6500 Scintillation counter To prepare samples for electron microscopy 470

proteoliposomes were mixed with a reaction buffer containing 20 mM Tris-HCl pH 75 100 471

mM sodium chloride 10 glycerol 20 mM manganese chloride and 3 mM UDP-Glc and 472

incubated at 37degC for 3 h which was followed by treatment with 01 triton X-100 473

474

Kinetic analysis 475

Reactions were performed with 20 mM MnCl2 and 0 to 35 mM UDP-Glc A fourfold 476

concentrated stock solution of 20 mM UDP-Glc and 10 μCi UDP-[3H]-Glc were diluted to the 477

final substrate concentration required in each reaction After incubation for 30 min the reactions 478


- 18 -

were treated as described above Untransformed data were fit to the Michaelis-Menton equation 479

to extract parameter values 480

481

Transmission Electron Microscopy (TEM) 482

400 mesh Glider Copper grids (Ted Pela) were coated by evaporation of carbon using Denton 483

Vacuum 502B carbon coater 35 μL of prepared sample was loaded onto a glow-discharged grid 484

incubated for 1 min and negatively stained with 10 serial drops of 075 uranyl formate on a 485

parafilm TEM images were taken using an FEI Tecnai 12 Spirit BioTWIN TEM [FEI 120 kV 486

63 mm spherical aberration (Cs) 56 K magnification 4 K times 4 K Eagle CCD camera located at 487

the Huck Institutes of the Life Sciences Penn State University] 488

489

Cryo-EM analysis 490

To measure the width of cellulose microfibrils in vitro cellulose microfibril products were 491

applied to Cryo EM analysis as described (Grassucci et al 2008) Never-dried sample was 492

loaded on a QUANTIFOIL holey carbon grid (300 mesh Ted Pella) blotted for 3 sec and 493

vitrified with a Vitrobot (FEI at the Huck Institute of the Life Sciences Penn State University) 494

495

Cellulase sensitivity assay 496

For assays of incorporation of radioactive UDP glucose the in vitro products were incubated 497

with 10 U of β-14-glucanases or β-13-glucanases (Megazyme) at 37 degC for 1 h For TEM 498

observation in vitro produced cellulose microfibrils were incubated with a total of 150 ng highly 499

purified cellulase mix containing Cel6A Cel7A and Cel7B proteins (kindly provided by Giridhar 500

Poosarla Penn State University) which was incubated at 50 degC Sample was taken for negative 501

staining 502

503

Linkage analysis 504

In vitro products were pre-treated for linkage analysis as described (Purushotham et al 505

2016) Briefly 2 SDS and 1 mgml proteinase K were used to eliminate proteins followed by 506

washing twice with water treatment with hexane and then freeze-drying Dried samples were 507

treated with chloroformmethanol followed by washing with 70 ethanol and then a solution 508

containing 50 mM Tris-HCl 2 SDS 10 mM Na-EDTA 40 mM β-mercaptoethanol was used 509


- 19 -

to remove residual proteins followed by washing 3 times with 70 ethanol To eliminate 510

Pichia-derived glycogen-like polysaccharide the samples were subjected to α-amylase and 511

amyloglucosidase and then washed with 70 ethanol Subsequently samples were freeze-dried 512

These treatments greatly reduced the mass (from 10 to 2 mg for wild-type and 33 to 42 mg for 513

PpCesA5(D782N) The prepared samples were permethylated to alditol acetates and analyzed by 514

GCEI-MS following the method previously described (Omadjela et al 2013) 515

516

Image analysis 517

All image analyses were performed using ImageJ (National Institute of Health) In order to 518

measure areas occupied by microfibrils the Set Scale option was used to set the real length the 519

Polygon selection tool was used to define areas along the microfibrils and the Measure tool was 520

used to measure area Measured areas were analyzed by mean and standard error For 521

measurement of thickness of individual microfibrils the lsquoStraight Linersquo tool was used to define 522

widths and the lsquoMeasurersquo tool was used for measurement of thickness 523

524

Mass spectrometry 525

For mass spectrometry analysis partially purified protein sample was separated by SDS-526

PAGE followed by coomassie staining The gel region corresponding to 110-140 kDa was cut 527

out and incubated with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)08 (wv) 528

ammonium bicarbonate at 60degC for 10 min followed by washing with 100 mM iodoacetamide 529

(IAA)08 (wv) ammonium bicarbonate at 37degC for 15 min MS spectra were taken from 60 530

minute gradient from an Eksigent NanoLC-Ultra-2D Plus and Eksigent LC through a 200 microm x 531

05 mm Chrom XP C18-CL 3 microm 120 Aring Trap Column and elution through a 75 microm x 15 cm 532

NanoLCMS C18-CL 26 microm 120 Aring Nano LC Column Sciex 5600 TripleTOF settings were 533

Parent scan acquired for 250 msec and then up to 50 MSMS spectra were acquired over 25 534

seconds for a total cycle time of 28 sec Gas 1 (Nitrogen) = 7 and Gas 3 (Nitrogen)= 25 Mass 535

spectrometry analysis was performed by the Proteomics and Mass Spectrometry Core Facility of 536

Penn State Hershey 537

538

Supplemental materials 539

Figure S1 ndash Diagram of PpCesA5 540


- 20 -

Figure S2 ndash Purification of heterologously expressed PpCesA5 from Pichia 541

Figure S3 ndash Purification of PpCesA5(D782N) 542

Figure S4 ndash Biochemical activity of PpCesA5(D782N) protein 543

Figure S5 ndash Proteoliposomes bearing PpCesA5(D782N) produce no microfibrils 544

Figure S6 ndash TEM analysis of in vitro products from disrupted proteoliposomes bearing PpCesA5 545

Figure S7 ndash TEM analysis of in vitro products from proteoliposomes bearing PpCesA5 546

Figure S8 ndash Cryo EM image of cellulose microfibrils 547

Figure S9 ndash Fragmentation spectra from electron-impact mass spectrometry of the permethylated 548

alditol acetates of the in vitro generated polysaccharide 549

Figure S10 ndash Linkage analysis of in vitro products of proteoliposomes bearing PpCesA5(D782N) 550

Supplemental Table S1 ndash List of proteins identified by mass spectrometry 551

552

Acknowledgments 553

We thank Missy Hazen and John Cantolina (Huck Institutes of the Life Sciences Penn State 554

University) for technical help with TEM and Giridhar Poosarla (Penn State University) for 555

providing Cel6A Cel7A and Cel7B proteins 556

557

Figure legends 558 559

Figure 1 Partial purification of heterologously expressed PpCesA5 from Pichia Membrane 560

proteins were isolated from P pastoris expressing PpCesA512xHis and purified using TALON 561

(cobalt) resin A SDS-PAGE gel stained with coomassie Lane 1 total protein extract Lane 2 562

non-membrane proteins Lane 3 membrane proteins Lane 4 flow through Lane 5 washing 563

fraction 1 (20 mM imidazole) Lane 6 washing fraction 2 (40 mM imidazole) Lane 7 washing 564

fraction 3 (60 mM imidazole) Lane 8 washing fraction 4 (80 mM imidazole) Lane 9 Elution 565

(350 mM imidazole) B Immunoblot of gel as in (A) using PpCesA5 specific antibody 1 The 566

arrows point to bands of mass expected for full length PpCesA5 See Supplemental Figure S1 for 567

epitope location 568

569

Figure 2 Functional characterization of PpCesA5 reconstituted into liposomes A Partially 570

purified PpCesA5 protein was reconstituted into liposomes using yeast total lipids and detergent 571


- 21 -

removal by BioBeadsreg

and then assayed for function by incubation with UDP-Glc and UDP-572

[3H]-Glc in the presence of the indicated cations After stopping reactions by the addition of 573

Triton X-100 the product was applied to descending paper chromatography from which 574

insoluble material at the origin was evaluated in a scintillation counter Error bars represent 575

standard deviation (n=3) B Time course C Kinetic analysis Michaelis-Menten parameter 576

estimates and 95 confidence intervals KM=137 (102179) microM Vmax=103 (96111) relative 577

units D Treatments with β-14-glucanase (cellulase) or β-13-glucanase (callase) 578

579

Figure 3 PpCesA5 in proteoliposomes produces microfibrils A-F Proteoliposomes bearing 580

PpCesA5 were incubated with UDP-Glc during which samples were taken at designated time 581

points for imaging by negative stain TEM Representative random images are shown Arrows 582

indicate microfibrils magnified in insets for panels B and C G Negative control ndash 583

representative image for a reaction incubated for 180 min without UDP-Glc Scale bars - 100 nm 584

H Plots of the mean and standard error for the areas occupied by microfibrils in 40 random 585

images NC negative control 586

587

Figure 4 Microfibrils are sensitive to cellulose-specific glucosidases To examine if the 588

microfibrils were made of cellulose in vitro products were subjected to cellulase treatment and 589

sampled at the designated time points by negative staining Randomly taken TEM images were 590

analyzed for areas occupied by fibrils A-F Representative images for reaction times of 0 5 10 591

30 45 and 60 min G Negative control ndash representative image for a reaction incubated for 60 592

min without cellulase Arrows indicate microfibrils Arrowheads in A and G indicate bundled 593

microfibrils Scale bars - 100 nm H Plots of the mean values of the areas occupied by 594

microfibrils Gray bars and white bars represent total microfibrils and bundled microfibrils 595

respectively Error bars indicate standard errors (n=40) NC negative control 596

597

Figure 5 Microfibrils contain 1-4 linked glucose In vitro products were pretreated with 2 598

SDS and proteinase K followed by α-amylase and amyloglucosidase to remove Pichia-derived 599

α-14-glucan chains Thereafter the sample was permethylated to alditol acetates and then 600

subjected to GCMS analysis The resulting spectra are compared with reference peaks Identities 601


- 22 -

of each peak are marked In particular terminal-Glc (t-Glc) and 4-linked Glc (4-Glc) were 602

detected at 9348 min and 19059 min respectively 603

604

Figure 6 Cellulose microfibrils are produced from PpCesA5 in proteoliposomes and coalesce 605

into higher order macrofibrils Proteoliposomes bearing PpCesA5 were incubated with UDP-Glc 606

followed by negative staining and TEM analysis Arrows indicate where cellulose microfibrils 607

were attached to proteoliposomes (A and B) Arrowheads indicate where microfibrils coalesced 608

into macrofibrils (C-F) Scale bars - 100 nm 609

610

Figure 7 Celluose microfibrils from PttCesA8-proteoliposomes coalesce into higher order 611

macrofibrils Proteoliposomes bearing PttCesA8 were incubated with UDP-Glc followed by 612

negative staining and TEM analysis Arrows indicate where cellulose microfibrils coalesced into 613

macrofibrils Scale bars - 100 nm 614

615

616

Literature Cited 617

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reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787-796 619

Arioli T Peng L Betzner AS Burn J Wittke W Herth W Camilleri C Hofte H Plazinski 620

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of cellulose biosynthesis in Arabidopsis Science 279 717-720 622

Atalla RH Vanderhart DL (1984) Native cellulose a composite of two distinct crystalline 623

forms Science 223 283-285 624

Augimeri RV Varley AJ Strap JL (2015) Establishing a Role for Bacterial Cellulose in 625

Environmental Interactions Lessons Learned from Diverse Biofilm-Producing 626

Proteobacteria Front Microbiol 6 1282 627

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222 629

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1896-1907 632

Basu S Omadjela O Zimmer J Catchmark JM (2017) Impact of plant matrix 633

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nuclear magnetic resonance spectroscopy of tunicin an animal cellulose 637

Macromolecules 22 1615-1617 638


- 23 -

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(2006) Interactions between MUR10CesA7-dependent secondary cellulose biosynthesis 640

and primary cell wall structure Plant Physiol 142 1353-1363 641

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Cano-Delgado A Penfield S Smith C Catley M Bevan M (2003) Reduced cellulose 645

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351-362 647

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301-312 661

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Gonneau M Desprez T Guillot A Vernhettes S Hofte H (2014) Catalytic subunit 668

stoichiometry within the cellulose synthase complex Plant Physiol 166 1709-1712 669

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3 330-339 675

Ha MA Apperley DC Evans BW Huxham IM Jardine WG Vietor RJ Reis D Vian B 676

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and quince Plant J 16 183-190 678

Hill JL Jr Hammudi MB Tien M (2014) The Arabidopsis cellulose synthase complex a 679

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4842 681

Him JL Pelosi L Chanzy H Putaux JL Bulone V (2001) Biosynthesis of (1--gt3)-beta-D-682

glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana 683

Eur J Biochem 268 4628-4638 684


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John RP Anisha GS Nampoothiri KM Pandey A (2011) Micro and macroalgal biomass a 685

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Kimura S Laosinchai W Itoh T Cui X Linder CR Brown RM Jr (1999) Immunogold 687

labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant vigna 688

angularis Plant Cell 11 2075-2086 689

Kurek I Kawagoe Y Jacob-Wilk D Doblin M Delmer D (2002) Dimerization of cotton fiber 690

cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains 691

Proc Natl Acad Sci U S A 99 11109-11114 692

Marga F Grandbois M Cosgrove DJ Baskin TI (2005) Cell wall extension results in the 693

coordinate separation of parallel microfibrils evidence from scanning electron 694

microscopy and atomic force microscopy Plant J 43 181-190 695

McFarlane HE Doring A Persson S (2014) The cell biology of cellulose synthesis Annu Rev 696

Plant Biol 65 69-94 697

McNamara JT Morgan JL Zimmer J (2015) A molecular description of cellulose 698

biosynthesis Annu Rev Biochem 84 895-921 699

Mendu V Stork J Harris D DeBolt S (2011) Cellulose synthesis in two secondary cell wall 700

processes in a single cell type Plant Signal Behav 6 1638-1643 701

Morgan JL McNamara JT Zimmer J (2014) Mechanism of activation of bacterial cellulose 702

synthase by cyclic di-GMP Nat Struct Mol Biol 21 489-496 703

Newman RH Hill SJ Harris PJ (2013) Wide-angle x-ray scattering and solid-state nuclear 704

magnetic resonance data combined to test models for cellulose microfibrils in mung bean 705

cell walls Plant Physiol 163 1558-1567 706

Nishiyama Y Langan P Chanzy H (2002) Crystal structure and hydrogen-bonding system in 707

cellulose Ibeta from synchrotron X-ray and neutron fiber diffraction J Am Chem Soc 124 708

9074-9082 709

Nishiyama Y Sugiyama J Chanzy H Langan P (2003) Crystal structure and hydrogen 710

bonding system in cellulose I(alpha) from synchrotron X-ray and neutron fiber diffraction 711

J Am Chem Soc 125 14300-14306 712

Nixon BT Mansouri K Singh A Du J Davis JK Lee JG Slabaugh E Vandavasi VG 713

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Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the 715

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Olek AT Rayon C Makowski L Kim HR Ciesielski P Badger J Paul LN Ghosh S 717

Kihara D Crowley M Himmel ME Bolin JT Carpita NC (2014) The structure of the 718

catalytic domain of a plant cellulose synthase and its assembly into dimers Plant Cell 26 719

2996-3009 720

Omadjela O Narahari A Strumillo J Melida H Mazur O Bulone V Zimmer J (2013) 721

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase 722

sufficient for in vitro cellulose synthesis Proc Natl Acad Sci U S A 110 17856-17861 723

Pauly M Keegstra K (2008) Cell-wall carbohydrates and their modification as a resource for 724

biofuels Plant J 54 559-568 725

Persson S Paredez A Carroll A Palsdottir H Doblin M Poindexter P Khitrov N Auer M 726

Somerville CR (2007) Genetic evidence for three unique components in primary cell-727

wall cellulose synthase complexes in Arabidopsis Proc Natl Acad Sci U S A 104 15566-728

15571 729


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Stengel DB (2011) Evolution and diversity of plant cell walls from algae to flowering 731

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Purushotham P Cho SH Diaz-Moreno SM Kumar M Nixon BT Bulone V Zimmer J 733

(2016) A single heterologously expressed plant cellulose synthase isoform is sufficient 734

for cellulose microfibril formation in vitro Proc Natl Acad Sci U S A 113 11360-11365 735

Roberts AW Bushoven JT (2007) The cellulose synthase (CESA) gene superfamily of the 736

moss Physcomitrella patens Plant Mol Biol 63 207-219 737

Roberts AW Roberts EM Haigler CH (2012) Moss cell walls structure and biosynthesis 738

Front Plant Sci 3 166 739

Saxena IM Brown Jr RM (1997) Identification of cellulose synthase(s) in higher plants 740

Sequence analysis of processive szlig-glycosyltransferases with the common motif D D 741

D35Q(RQ)XRW Cellulose 4 33-49 742

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1148 745

Sethaphong L Haigler CH Kubicki JD Zimmer J Bonetta D DeBolt S Yingling YG 746

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7512-7517 748

Slabaugh E Davis JK Haigler CH Yingling YG Zimmer J (2014) Cellulose synthases new 749

insights from crystallography and modeling Trends Plant Sci 19 99-106 750

Somerville C (2006) Cellulose synthesis in higher plants Annu Rev Cell Dev Biol 22 53-78 751

Szymanski DB Cosgrove DJ (2009) Dynamic coordination of cytoskeletal and cell wall 752

systems during plant cell morphogenesis Curr Biol 19 R800-811 753

Taylor NG Laurie S Turner SR (2000) Multiple cellulose synthase catalytic subunits are 754

required for cellulose synthesis in Arabidopsis Plant Cell 12 2529-2540 755

Thomas LH Forsyth VT Sturcova A Kennedy CJ May RP Altaner CM Apperley DC 756

Wess TJ Jarvis MC (2013) Structure of cellulose microfibrils in primary cell walls 757

from collenchyma Plant Physiol 161 465-476 758

Vandavasi VG Putnam DK Zhang Q Petridis L Heller WT Nixon BT Haigler CH 759

Kalluri U Coates L Langan P Smith JC Meiler J ONeill H (2016) A Structural 760

Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex 761

Evidence for CESA Trimers Plant Physiol 170 123-135 762

Wise HZ Saxena IM Brown RM (2011) Isolation and characterization of the cellulose 763

synthase genes PpCesA6 and PpCesA7 in Physcomitrella patens Cellulose 18 371-384 764

Zhang T Zheng Y Cosgrove DJ (2016) Spatial organization of cellulose microfibrils and 765

matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic 766

force microscopy Plant J 85 179-192 767

768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B

Figure 1 Partial purification of heterologously expressed PpCesA5 from Pichia Membrane proteins were isolated from P pastoris expressing PpCesA512xHis and purified using TALON (cobalt) resin A SDS-PAGE gel stained with coomassie Lane 1 total protein extract Lane 2 non-membrane proteins Lane 3 membrane proteins Lane 4 flow through Lane 5 washing fraction 1 (20 mM imidazole) Lane 6 washing fraction 2 (40 mM imidazole) Lane 7 washing fraction 3 (60 mM imidazole) Lane 8 wash-ing fraction 4 (80 mM imidazole) Lane 9 Elution (350 mM imidazole) B Immunoblot of gel as in (A) using PpCesA5 specific antibody 1 The arrow indicates the band corresponding to PpCesA5 See Supplemental Figure S1 for epitope location


Figure 2

Figure 2 Functional characterization of PpCesA5 reconstituted into liposomes A Partially purified PpCesA5 protein was reconstituted into liposomes using yeast total lipids and detergent removal by BioBeadsreg and then assayed for function by incubation with UDP-Glc and UDP-[ H]-Glc in the pres-ence of the indicated cations After stopping reactions by the addition of Triton X-100 the product was applied to descending paper chromatography from which insoluble material at the origin was evaluated in a scintillation counter Error bars represent standard deviation (n=3) B Time course C Kinetic analysis Michaelis-Menten parameter estimates and 95 confidence intervals KM=137 (102179) microM Vmax=103 (96111) relative units D Treatments with β-14-glucanase (cellulase) or β-13-glucanase (callase)

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Figure 3

Figure 3 PpCesA5 in proteoliposomes produces microfibrils A-F Proteoliposomes bearing PpCesA5 were incubated with UDP-Glc during which samples were taken at designated time points for imaging by negative stain TEM Representative random images are shown G Negative control ndash representa-tive image for a reaction incubated for 180 min without UDP-Glc Scale bars - 100 nm H Plots of the mean and standard error for the areas occupied by microfibrils in 40 random images NC negative con-trol

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Figure 4

Figure 4 Microfibrils are sensitive to cellulose-specific glucosidases To examine if the microfibrils were made of cellulose in vitro products were subjected to cellulase treatment and sampled at the des-ignated time points by negative staining Randomly taken TEM images were analyzed for areas occu-pied by fibrils A-F Representative images for reaction times of 0 5 10 30 45 and 60 min G Negative control ndash representative image for a reaction incubated for 60 min without cellulase Scale bars - 100 nm H Plots of the mean values of the areas occupied by microfibrils Error bars indicate standard errors (n=40) NC negative control

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Figure 5

Figure 5 Microfibrils contain 1-4 linked glucose In vitro products were pretreated with 2 SDS and proteinase K followed by α-amylase and amyloglucosidase to remove Pichia-derived α-14-glucan chains Thereafter the sample was permethylated to alditol acetates and then subjected to GCMS analysis The resulting spectra are compared with reference peaks Identities of each peak are marked In particular terminal-Glc (t-Glc) and 4-linked Glc (4-Glc) were detected at 9348 min and 19059 min respectively


Figure 6

Figure 6 Cellulose microfibrils are produced from PpCesA5 in proteoliposomes Proteoliposomes bearing PpCesA5 were incubated with UDP-Glc followed by negative staining and TEM analysis Arrows indicate where cellulose microfibrils were attached to proteoliposomes Scale bars - 100 nm

A

B


Figure 7

Figure 7 Celluose microfibrils from PpCesA5 proteoliposomes coalesce into higher order macrofibrils Proteoliposomes bearing PpCesA5 were incubated with UDP-Glc followed by negative staining and TEM analysis Arrows indicate where cellulose microfibrils coalesced into macrofibrils Scale bars - 100 nm

A B

DC


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Parsed Citations

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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Parsed Citations

- 2 -

Synthesis and self-assembly of cellulose microfibrils from reconstituted cellulose synthase 24

25

Sung Hyun Cho1 Pallinti Purushotham

2 Chao Fang

3 Cassandra Maranas

4 Sara M Diacuteaz-26

Moreno5 Vincent Bulone

56 Jochen Zimmer

2 Manish Kumar

3 B Tracy Nixon

1 27

28

1Department of Biochemistry and Molecular Biology The Pennsylvania State University 29


2Department of Molecular Physiology and Biological Physics University of Virginia School of 31

Medicine 480 Ray C Hunt Dr Charlottesville VA 22908 USA 32

3Department of Chemical Engineering The Pennsylvania State University University Park PA 33

16802 USA 34

4Department of Chemical Engineering University of Washington Seattle WA 98105 35

5Division of Glycoscience School of Biotechnology Royal Institute of Technology (KTH) 36

Stockholm SE-10691 Sweden 37

6Australian Research Council Centre of Excellence in Plant Cell Walls School of Agriculture 38

Food and Wine University of Adelaide Waite Campus Urrbrae 5064 South Australia 39

Australia 40

41

One Sentence Summary 42

Liposome-reconstituted heterologously expressed cellulose synthases that contribute to 43

primarysecondary plant cell walls synthesized glucan chains that assembled into cellulose 44

microfibrils45


- 3 -

Footnotes 46





linkage analysis 51

52





57


59


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Abstract 60





















81


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Introduction 82































- 6 -









materials 120














2016) A Zn2+











- 7 -

































- 8 -

174

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176
























200






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Mg2+

Mn2+

and Zn2+

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found that Mn2+


























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- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








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staining 502

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- 19 -







516

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524














538




- 20 -












552

Acknowledgments 553




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579








587










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- 22 -



604






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- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






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- 24 -













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- 3 -

Footnotes 46





linkage analysis 51

52





57


59


- 4 -

Abstract 60





















81


- 5 -

Introduction 82































- 6 -









materials 120














2016) A Zn2+











- 7 -

































- 8 -

174

Results 175

176
























200






- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

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Figure 3


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Article File

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Parsed Citations

- 4 -

Abstract 60





















81


- 5 -

Introduction 82































- 6 -









materials 120














2016) A Zn2+











- 7 -

































- 8 -

174

Results 175

176
























200






- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

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15 30 45 60 90 120 150 180 210

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A B

C

0

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2000

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4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

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(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

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Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 5 -

Introduction 82































- 6 -









materials 120














2016) A Zn2+











- 7 -

































- 8 -

174

Results 175

176
























200






- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 6 -









materials 120














2016) A Zn2+











- 7 -

































- 8 -

174

Results 175

176
























200






- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 7 -

































- 8 -

174

Results 175

176
























200






- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 8 -

174

Results 175

176
























200






- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 9 -







Mg2+

Mn2+

and Zn2+

We 210

found that Mn2+


























233


- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 10 -































263


- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 11 -



















282

Discussion 283







macrofibrilsiv

or bundlesiv

) 289










- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 12 -

























microfibrilsiv










- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 13 -


























348










- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 14 -









-binding 363





368

Conclusions 369




and 372

macrofibrilsiv

or bundlesiv











383


385



- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 15 -
























409










- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 16 -




















437











448


- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 17 -










458

Activity assay 459















474






- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 18 -



481








489






495







staining 502

503








- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 19 -







516

Image analysis 517







524














538




- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 20 -












552

Acknowledgments 553




557











569




- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 21 -









579








587










597






- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 22 -



604






610





615

616













222 629



1896-1907 632








- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 23 -









351-362 647














301-312 661










patens Planta 235 1355-1367 672



3 330-339 675






4842 681



Eur J Biochem 268 4628-4638 684


- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 24 -













Plant Biol 65 69-94 697












9074-9082 709



J Am Chem Soc 125 14300-14306 712








2996-3009 720









15571 729


- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

- 25 -
















1148 745



7512-7517 748




















768

769


Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Figure 1

260

1401007050

40

(Kda)1 2 3 4 5 6 7 8 9

A

260

1401007050

40

(Kda)B



Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

rofb

ril a

reas

(nm

2 )

Time (min)



A B C D

E F G HH (x10 3)


Figure 4


0

5

10

15

20

25

0 5 1015 3045 60NCMic

rofib

ril a

reas

(nm

2 )


A B C D



(x103)


Figure 5



Figure 6


A

B


Figure 7


A B

DC
















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Figure 2


0

1000

2000

3000

4000

5000

6000


DPM

Cations

0

1000

2000

3000

4000

5000

15 30 45 60 90 120 150 180 210

DPM

Time (min)

A B

C

0

1000

2000

3000

4000

5000

6000


DPM

D

Rel

ativ

e ac

tivity

()

00 05 10 15 20 25 30 35UDP-Glc (mM)

120

100

80

60

40

20

0

2+ 2+ 2+ 2+ 2+ 2+

3


Figure 3


01234567

0 5 10 15 30 45 60 180

NC Mic

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2 In vitro - Plant Physiology€¦ · 32 Medicine, 480 Ray C. Hunt Dr., Charlottesville, VA 22908,...

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