Cultivars - ACS Publications - American Chemical Society

Jul 27, 2016 - In this work, the spatial distribution of AX and BG in the endosperm of mature grains was established for nine wheat varieties and eigh...
0 downloads 0 Views 2MB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Mass Spectrometric Imaging of Wheat (Triticum spp.) and Barley (Hordeum vulgare L.) Cultivars: Distribution of Major Cell Wall Polysaccharides According to Their Main Structural Features Dusan Velickovic, Luc Saulnier, Margot Lhomme, Aurélie Damond, Fabienne Guillon, and Hélène Rogniaux J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02047 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Mass Spectrometric Imaging of Wheat (Triticum spp.) and Barley

2

(Hordeum vulgare L.) Cultivars: Distribution of Major Cell Wall

3

Polysaccharides According to Their Main Structural Features

4

Dušan Veličković, Luc Saulnier, Margot Lhomme, Aurélie Damond, Fabienne Guillon,

5

Hélène Rogniaux *

6

INRA, UR1268 Biopolymers Interactions Assemblies F-44316 NANTES, France.

7

* To whom correspondence should be addressed. e-mail: [email protected],

8

Tel.: +33 (0)2 40 67 50 34, Fax: +33 (0)2 40 67 50 25

9

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 32

10

Abstract

11

Arabinoxylans (AX) and (1→3), (1→4)-β-glucans (BG) are the main components of cereal

12

cell walls and influence many aspects of their end uses. Important variations in the

13

composition and structure of these polysaccharides have been reported among cereals and

14

cultivars of a given species. In this work, the spatial distribution of AX and BG in the

15

endosperm of mature grains was established for nine wheat varieties and eight barley varieties

16

using enzymatically-assisted mass spectrometry imaging (MSI). Important structural features

17

of the AX and BG polymers that were previously shown to influence their physico-chemical

18

properties were assessed. Differences in the distribution of AX and BG structures were

19

observed, both within the endosperm of a given cultivar and between wheat and barley

20

cultivars. This study provides a unique picture of the structural heterogeneity of AX and BG

21

polysaccharides at the scale of the whole endosperm in a series of wheat and barley cultivars.

22

Thus, it can participate meaningfully in a strategy aiming at understanding the structure-

23

function relationships of these two polymers.

24

Key words: arabinoxylans, beta glucans, wheat (Triticum), barley (Hordeum vulgare), cell

25

wall, MALDI-MS imaging, plant, polysaccharides.

2 ACS Paragon Plus Environment

Page 3 of 32

Journal of Agricultural and Food Chemistry

26

Introduction

27

A distinctive feature of cereal grain cell walls is the widespread adoption of heteroxylans

28

(arabinoxylans, AX) and (1→3, 1→4)-β-glucans (BG) as the major non-cellulosic

29

polysaccharides of the starchy endosperm.1 These non-cellulosic polysaccharides significantly

30

influence the quality of the grain and its end-uses, including food processing, livestock feed,

31

and alcohol production.2 They also make an important contribution to the daily intake of

32

dietary fibers and its associated health benefits, thus leading breeders to select cereal varieties

33

based on the level of these non-cellulosic wall polysaccharides.

34

However, this is a difficult task because there is considerable variability in the composition of

35

the grain cell wall between cereal species and among varieties of the same species.3-5 This

36

variability is likely driven by genetics,6,

37

understood. For example, the starchy endosperm cell walls of rye and wheat grains are

38

characterized by a high AX content relative to the amount of BG. While for barley and oats,

39

the endosperm cell walls are predominantly composed of BG.1 Additionally, the amount of

40

cell wall AX in the starchy endosperm is higher in rye than in wheat and in barley than in

41

oats. For example, the total AX level in white flours ranges from 1.35-2.75% of dry matter

42

(dm) for wheat species, from 3.11-4.31% dm for rye, from 0.97-1.26% dm for oats and from

43

1.4-2.2% dm for barley.8-11

44

The structural features of AX and BG in the starchy endosperm of cereal grains vary

45

according to species, cultivar, developmental stage and cell position in the tissue.12 The

46

primary structure of AX found in the endosperms of wheat, rye, and barley is similar. AX is

47

formed of a β-(1,4)-D-xylan backbone with a single arabinose unit present on the main chain

48

as a mono-substitution on position O-3 (mXyl3), or di-substitution on positions O-2 and O-3

49

(dXyl) of the xylose residues (Figure 1A). Mono-substitution on O-2 (mXyl2) is rare in wheat

50

and rye but represents a significant proportion of AX in barley. The ratio between un-, mono-,

7

and its biological significance is far from

3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 32

51

and di-substituted xylose residues is approximately 65:15:20 for wheat, 55:35:10 for rye and

52

65:10:25 for barley. 13 The arabinose to xylose ratio (A:X) is often used to characterize the

53

structure of AX; in wheat, rye or barley. A typical A:X ratio is approximately 0.5–0.6.12

54

However, similar A:X ratios do not always reflect the same arabinose distribution on the

55

xylan backbone. Actually, for the same A:X ratio, rye AX has less unsubstituted xylose

56

residues than wheat and barley AX, due to a higher proportion of mXyl. AX structure is also

57

impacted by spatial and temporal changes in arabinose substitution. This has been studied,

58

especially in wheat, by several groups using microscopy, vibrational micro-spectroscopy, or

59

mass spectrometry imaging.14-16 Briefly, a gradient of AX substitution was reported across the

60

wheat grain with the less substituted AX found in the peripheral cells of the endosperm.

61

During grain development, the level of arabinose substitution also varies, with the substitution

62

decreasing upon maturation.16 Changes in the distribution of arabinose modify chain-chain

63

interactions. Studies using films made from differently structured AX showed changes in

64

water motion and mechanical properties dependent on the number of arabinose substitutions;

65

higher water diffusion was observed with highly substituted AX compared to less substituted

66

AX.12,

67

mechanical properties of the cell wall and consequently the development and final quality of

68

the grain.18

69

BG polymer is composed of a linear chain of β-D-glucosyl residues linked by (1→4) and

70

(1→3) linkages. The polysaccharide chain is predominantly (90%) comprised of cellotriosyl

71

(degree of polymerization three: DP3, abbreviated as BG3) and cellotetraosyl units (degree of

72

polymerization four: DP4, abbreviated as BG4), linked by β-(1→3) linkages (Figure 1B).

73

Approximately 10% of the polysaccharide is comprised of longer chains of adjacent β-(1→4)

74

linked glucose units.1 The DP3 and DP4 units are arranged randomly along the chain. Yet,

75

this arrangement, and hence the distribution of the β-(1→3) linkages, impacts the solubility

17

Thus, the fine structural tuning of AX probably impacts the hydration and

4 ACS Paragon Plus Environment

Page 5 of 32

Journal of Agricultural and Food Chemistry

76

and aggregation properties of the polymer by altering chain-chain interactions. High ratios of

77

DP3:DP4 and large amounts of longer cellodextrin fragments (DP5-DP9) in BG chains have

78

been associated with the decreased solubility or extractability of these polysaccharides from

79

cereal grains 19 and is reflected in their digestibility. 20 BG structure varies among cereals: the

80

molar ratio of DP3:DP4 units is in the range of 3.0-4.5 for wheat, 1.8-3.5 for barley, 1.9-3.0

81

for rye and 1.5-2.3 for oats.21,

82

spatial or temporal changes in the DP3:DP4 ratio, it can be presumed that it is significantly

83

heterogeneous throughout the grain and among species.14, 23, 24

84

Given the major contribution of cereals to the daily diet of billions of people and their

85

economic importance, there is a compelling need to better describe and anticipate the

86

structural variability of their endosperm cell wall components. There have been constant

87

efforts to develop reliable antibodies and/or carbohydrate binding modules to detect the

88

spatial heterogeneity of cell wall polysaccharides in cereal grains.25-27 Vibrational

89

spectroscopy has been successfully used to monitor both temporal and spatial-related

90

structural changes in these polymers.5,

91

method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS

92

imaging, or MALDI-MSI) to probe AX and BG in the developing wheat endosperm.14 After

93

mounting the tissue onto a conductive glass plate and applying the MALDI matrix, the MS

94

instrument captures a series of mass spectra, each of which represent the mass profile of a

95

laser beam-irradiated region of the sample. Ion intensities are then plotted on a coordinate

96

system matching the relative location of each spectrum to create a molecular image of the

97

tissue surface.30 MALDI-MSI thus couples spatial information at 20-100 µm resolution with

98

the abundant chemical and structural information provided by MS.14, 31-34

99

In the present study, we applied MALDI-MSI to study the endosperm cell walls of several

100

wheat and barley varieties. The spatial distribution of AX and BG oligosaccharides (AXOS

22

Although little information is currently available on the

15, 28, 29

Recently, our group proposed an imaging

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 32

101

and BGOS) released after enzymatic degradation of the walls was established across the

102

wheat and barley grains. In particular, some structural features of importance for the physico-

103

chemical properties of these polymers were monitored and imaged throughout the grain such

104

as the relative amount of mono- versus di-substituted AX and the relative amount of DP3 and

105

DP4 released from the BG.

106

Materials and Methods

107

Wheat samples

108

French cultivars of wheat (Triticum aestivum L.) grown in Ménétrol, France (harvested in

109

2001) were provided at maturity by Ulice (Riom, France) and were kept in closed 20 mL

110

plastic tubes in a storage room at 18-22 °C. The eight cultivars 'Aligre', 'Baltimore', 'Crousty',

111

'Magdalena', 'Mallaca', 'Sisley', 'Tamaro' and 'Thesee' were selected from a larger set of

112

samples on the basis of their different AX structures.3 AX contents of white flours ranged

113

from 1.7-2.7% dm, and the proportions of mono- and di-substituted AX varied.3 In addition,

114

the mature grains from the cultivar 'Recital' were used and were grown in a glasshouse under

115

conditions of natural day length at the INRA Station of Le Rheu, France in 2013).

116

Barley samples

117

Eight barley cultivars from the Healthgrain diversity screen were selected.10 Plants were

118

grown in Martonvasar, Hungary (harvested in 2005) and mature grains were kept in closed

119

plastic tubes in a storage room at 18-22 °C. Cultivars 'Plaisant' (subsequently referred to as

120

HGB2), 'Igri' (HGB3), 'Rastik' (HGB4), 'CFL93-149' (HGB5), 'CFL98-398' (HGB6), 'CFL98-

121

450' (HGB7), 'Erhard-Frederichen' (HGB8) and 'Morex' (HGB10) were analyzed. All were

122

hulled type except for 'Rastik' and CFL98-450, which were naked. BG contents in the grains

123

ranged from 3.7 up to 6.5% dm.10

124

Chemicals and reagents 6 ACS Paragon Plus Environment

Page 7 of 32

Journal of Agricultural and Food Chemistry

125

2,5-Dihydroxybenzoic acid (DHB) was purchased from Sigma-Aldrich Co (Saint Quentin

126

Fallavier, France). N, N‐Dimethylaniline (DMA) was obtained from Fisher Scientific (Fisher

127

Bioblock Scientific S.A, Illkirch, France). Xyloglucan heptasaccharide (XXXG), which was

128

used as an internal standard, was procured from Megazyme (Bray, Ireland). Purified

129

galactomannan digests (degree of polymerization (DP) of 3 to 9), which were used as mass

130

calibration standards for the MALDI-TOF instrument, were kindly provided by the

131

Laboratoire de Chimie des Substances Naturelles (Université de Limoges, France). NaCl was

132

purchased from Merck and CaCl2 was purchased from Carlo-Erba. Acetonitrile (MeCN),

133

ethanol (EtOH) and methanol (MeOH) were HPLC grade (Carlo‐Erba Reagents, Val de Reuil,

134

France). Ultrapure water was obtained from a Milli‐Q apparatus (Millipore SAS, Molsheim,

135

France).

136

Enzymes

137

Endoxylanase (EC 3.2.1.8) from Trichoderma viride was purchased from Megazyme

138

(Xylanase M1, Bray, Ireland). The specific activity of the enzyme preparation determined by

139

the supplier on WE-AX (40 ºC, pH 4.5) was 2,300 U/mL, and the optimum pH was 4.5-5.

140

Lichenase (endo-1,3(4)-β-D-glucanase, E.C. 3.2.1.73) from Bacillus subtilis was obtained

141

from Megazyme. The specific activity of the enzyme preparation determined by the supplier

142

on barley β-glucan (40 ºC, pH 6.5) was 1,000 U/mL, and the optimum pH was 6.5-7.0. α-

143

Amylase (E.C. 3.2.1.1) from porcine pancreas was purchased from Sigma-Aldrich. The

144

specific activity of the enzyme preparation was ≥10 U/mg solid, where one unit of enzyme

145

liberated 1 mg of maltose from starch in 3 min at pH 6.9 and 20 °C.

146

Sample preparation for MALDI-MS imaging: removal of starch

147

Thin tissue sections (60 µm) were prepared as previously described.14 Just before use, the

148

tissue sections were treated with α-amylase to remove the starch.35 Briefly, each tissue section

7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 32

149

was put in an Eppendorf tube filled with 0.5 mL of 1 mg/mL α-amylase solution in a 20 mM

150

Na-phosphate buffer with 2 mM NaCl and 0.25 mM CaCl2 at pH 6.9. After 24 h incubation at

151

40 °C, sections were rinsed in water and carefully mounted on indium tin oxide (ITO) glass

152

slides (Bruker Daltonics, Bremen, Germany, cat No 237001) using conductive carbon tape as

153

a support.

154

In situ digestion of cell wall polysaccharides

155

The enzymes (0.0014 U of xylanase or 0.0006 U of lichenase/mm2 of tissue) were

156

homogeneously applied to the tissue surface as fine droplets using an in-house-designed

157

spraying robot as previously described by Velickovic et al.14 After spraying, the tissues were

158

transferred to a closed container maintained at a relative humidity of 96.4 ± 0.4% with a

159

saturated solution of K2SO4 and then incubated at 40 ºC for 4 h. These conditions were

160

previously optimized by monitoring the release of the final degradation products (i.e., AX5

161

and AX6 for xylanase, and BG3 and BG4 for lichenase) and shown to be comparable to the

162

enzymatic digestion of ground cell wall material.35

163

MALDI matrix

164

An ionic dihydroxybenzoic acid/dimethylaniline matrix suitable for MALDI detection of

165

oligosaccharides

166

potassium adducts caused by the high concentrations of potassium in wheat

167

seeds 38 and instead, favor the sodium adducts on the mass spectra.

168

Application of the MALDI matrix for MSI

169

Application of the MALDI matrix was performed using an Image prep automatic vibration

170

vaporization system (Bruker Daltonics, Bremen, Germany). The matrix was applied in two

171

phases and the system settings were as follows: 1st Phase: 15 cycles: 12% spray power; 20%

172

spray modulation; 2 s spray time; 15 s incubation time; 30 s dry time. 2nd Phase: 40 cycles:

14, 36

was used. The matrix was prepared in 10 mM NaCl to reduce 37

and barley

8 ACS Paragon Plus Environment

Page 9 of 32

Journal of Agricultural and Food Chemistry

173

20% spray power; 25% spray modulation; 2 s spray time; 30 s incubation time; 60 s dry time.

174

A nitrogen gas flow (2x105 Pa) was provided during the entire procedure.

175

MSI Analysis

176

All MSI measurements were performed with an Autoflex-Speed MALDI‐TOF/TOF

177

spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355

178

nm, 1000 Hz) and controlled using the Flex Control 3.4 software package. The mass

179

spectrometer was operated with positive polarity in the reflectron mode, and spectra were

180

acquired in the range of m/z 500-2,000.

181

The laser raster size was set at 100 µm for quantitative experiments while a 50 µm laser raster

182

size was used for high-resolution MALDI-MSI. The signal was initially optimized by

183

manually adjusting the laser power and the number of laser shots. Full-scan MS spectra were

184

obtained by accumulating 200 laser shots per raster step, using the laser power that generated

185

the best signal-to-noise ratio. Under these conditions, it took approximately 30 min to

186

complete an image of a grain section at 100 µm resolution, while it took approximately 1 h at

187

50 µm resolution. Image acquisition was performed using the Flex Imaging 4.0 (Bruker

188

Daltonics) software package.

189

Quantification of released oligosaccharides and their variation between samples

190

For a given tissue section, the AX5:AX6 ratio was calculated by comparing the peak intensity

191

corresponding to the enzymatically released oligosaccharide AX5 (XA3XX according to

192

Faure et al,39 DP5; detected at m/z 701 as a sodiated cation [M+Na]+) in the average mass

193

spectrum of the tissue, to the intensity of the AX6 oligosaccharide (XA2+3XX according to

194

Faure et al,39 DP 6; detected at m/z 833, [M+Na]+ species). The BG3:BG4 ratio was similarly

195

determined, from the relative peak intensity of the m/z 527 (BG3, [M+Na]+ ion) to the m/z

196

689 (BG4, [M+Na]+ ion).

9 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 32

197

Statistical tests were performed to evaluate the variations of AX5:AX6 and BG3:BG4 ratios

198

within sections sampled across a single grain in the brush, center or germ regions as follows:

199

an F-test was first applied to check that the variances were equal between the data sets to be

200

compared. A t-test was used to determine significantly different sets of values. The result of

201

the t-test gives the probability that the means of the two compared data sets are the same. The

202

two data sets were considered significantly different when this probability was less than 5%.

203

Microscopy

204

The optical images that were overlapped with molecular images in the high-resolution

205

MALDI-MS imaging results were acquired on a Multizoom AZ100M microscope equipped

206

with a RGB DS-Ri1 camera 89 (Nikon, Japan). The microscope was equipped with 4 different

207

filter cubes: two with different UV 97 excitation (Filter UA : Excitation 325-375 nm,

208

Dichroic Mirror > 400 nm, Emission > 420 nm / Filter UB 98 : Ex 360-370 nm, DM > 380

209

nm, Em > 400 nm), one with blue excitation (Filter BL : Ex 450-490 nm, 99 DM > 505 nm,

210

Em > 515 nm) and the last with green excitation (Filter GR : Ex 510-560 nm, DM > 565 100

211

nm, Em > 593 nm). Samples were illuminated with a mercury lamp (Intensilight C-HGFIE,

212

Nikon, Japan). For each section, imaged were acquired under the four different spectral

213

conditions and images were merged using NIS software (Nikon). The exposure time for each

214

spectral condition was set on wheat cross-section to avoid saturation, especially on the

215

aleurone layer and the pericarp.

216

Results and Discussion

217

The distribution of AX and BG polymers in the endosperm of ten varieties of wheat and eight

218

varieties of barley were compared using MALDI-MS images of the grains’ cross sections.

219

Arabinoxylan oligosaccharides (AXOS) or beta glucan oligosaccharides (BGOS) with a DP

220

ranging from 3 to 10 were measured, following their release by enzymatic degradation of the

10 ACS Paragon Plus Environment

Page 11 of 32

Journal of Agricultural and Food Chemistry

221

cell walls with endoxylanase or endoglucanase (lichenase). More precisely, we monitored the

222

amount of enzymatically released AX5 (XA3XX of DP5) relative to AX6 (XA2+3XX of DP6),

223

and the relative amount of enzymatically released BG3 (i.e., BGOS with a DP of 3) relative to

224

BG4 (DP 4). In fact, both AX5:AX6 and BG3:BG4 ratios are related to important features of

225

the AX and BG polymers, which have been reported to impact their physico-chemical

226

properties. 18,

227

endoxylanase treatment has a mono-substitution on position O-3 (mXyl3) of the second

228

xylosyl residue, while AX6 is di-substituted on positions O-2 and O-3 (dXyl) of the same

229

xylose. Thus, the AX5:AX6 ratio indicates the extent of arabinosyl substitution of the AX

230

polymer, while the BG3:BG4 ratio after lichenase degradation (endo-1,3(4)- β-D-glucanase)

231

indicates β-(1→3) linkages in the BG polymer.

232

Qualification of the method

233

In a first step, experiments were performed to evaluate the reproducibility of the method and

234

the number of tissue sections needed to obtain a representative view of the wheat and barley

235

varieties by MALDI-MSI.

236

The technical reproducibility of MALDI-MSI was evaluated by measuring consecutive thin

237

cross-sections from the same grain (wheat, cv. 'Recital') after xylanase or lichenase treatment.

238

MALDI-MSI measurements cannot be repeated two times on the same tissue because after

239

one pass, the signal deteriorates. Thus, we found the best experimental procedure was to use

240

two consecutive sections (60 µm thickness) of a single grain, assuming that the variations in

241

AX5:AX6 or BG3:BG4 between these two sections was negligible. Two experiments were

242

then performed as follows: in the first experiment, the two consecutive sections were mounted

243

at the same (X,Y) position on two different plates. In another experiment, the consecutive

244

sections were placed at opposite corners of one glass plate. These two situations aimed to

245

determine both the variation due to the measurement (i.e., instrument instability), and the

20

As established previously by Ordaz-Ortiz et al, 3AX5 released after

11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 32

246

variation possibly arising from the glass-plate preparation (i.e., homogeneity of enzyme and

247

matrix deposition over the glass-plate). The variation of the measured AX5:AX6 ratio was

248

below 10% in the first experiment and 20% for the second one. The experimental error thus

249

comes mostly from inhomogeneity of the glass-plate preparation. This was considered to be

250

20% in the rest of the study.

251

Variability was then evaluated according to the positioning of the cross-section along the

252

same grain, for wheat (cv. 'Recital') and for barley (cv. CFL 98-450). A minimum of three

253

cross-sections (60 µm thickness for each) and up to six sections were sampled in the brush,

254

central, and germ regions of a grain. Tissue sections were then in situ hydrolyzed by xylanase

255

and lichenase and the AX5:AX6 and BG3:BG4 ratios were evaluated. The mean values of

256

AX5:AX6 and BG3:BG4 ratios were compared between the three regions. Statistically

257

significant differences were found between all sampled regions for BG3:BG4 in barley while

258

in wheat, significant differences were observed only for AX5:AX6 between the germ and the

259

other regions (brush or center). Because AX5:AX6 and BG3:BG4 ratios varied more in the

260

germ compared to the other regions for both wheat and barley, sections sampled from the

261

central part of the grains were used in the following experiments.

262

Lastly, we evaluated the inter-grain variability within the same variety. The typical amount of

263

starting material needed to investigate cell wall contents by biochemical methods is

264

approximately 10 g of ground material or 200-300 grains.3 Obviously MALDI-MSI cannot

265

handle such a large collection of grains. Thus, to evaluate the variance of the AX5:AX6 and

266

BG3:BG4 ratios among grains, a set of seven grains (and up to twelve) were selected, for one

267

variety of wheat (cv. 'Recital') and one variety of barley (cv. CFL98-450). Sections were

268

sampled from the center of each grain and treated with xylanase or lichenase. For barley, 60%

269

of the xylanase treated sections (n=10) exhibited AX5:AX6 ratios within +/- 20% of the mean

270

value, and 67% of the lichenase treated sections (n=12) exhibited BG3:BG4 ratios within +/12 ACS Paragon Plus Environment

Page 13 of 32

Journal of Agricultural and Food Chemistry

271

20% of the mean value. For wheat, 80% of the xylanase treated sections (n=13) had

272

AX5:AX6 ratios within +/- 20% of the mean value, and 75% of the lichenase treated sections

273

(n=8) had BG3:BG4 ratios within +/- 20% of the mean value. The 20% cut-off was chosen

274

because it corresponded to the previously determined technical error. From this investigation

275

it was deduced that, in the worst case, the probability that three randomly selected grains

276

within a given variety are all “outliers” (i.e., having a deviation from the mean value higher

277

than 20%) is 6.4%, i.e., 0.43. This probability falls to 2.5% with four grains.

278

Comparison of the structural features of AX and BG in wheat and barley varieties, and their

279

distribution in the grains

280

For the following experiments, a minimum of three grains (and up to six) were randomly

281

selected within the grain samples of each variety. One section sampled in the central part of

282

each grain was analyzed by MALDI-MSI, and imaged with a lateral resolution of 100 µm.

283

Note that due to tissue damage that occurred when applying some of the sections onto the

284

glass plates, not all sections could be analyzed. In rare cases, only two sections were used to

285

qualify the variety (five times out of the 36 experiments performed for the 18 varieties treated

286

with the two enzymes). When the standard deviation was above 20%, outlier values were

287

discarded in the series keeping at least three values to qualify the variety.

288

First, a global, quantitative interpretation of the images was performed. Figure 2 shows plots

289

of the average AX5:AX6 ratio as a function of the average BG3:BG4 ratio derived from the

290

MALDI-MSI measurements for the ten varieties of wheat and eight varieties of barley. The

291

different varieties are grouped by species along the AX5:AX6 axis. The AX5:AX6 ratio

292

suggests that barley varieties, on average, are richer in di-substituted AX than wheat varieties

293

(AX5:AX6 ratio is below 1 for all the barley varieties). Four of the ten varieties of wheat

294

('Sisley', 'Malacca', 'Aligre', 'Magdalena') have close AX5:AX6 ratios compared to some

295

barley varieties (in the range of 0.6-1.2). However, the six other wheat varieties display the 13 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 32

296

highest AX5:AX6 ratios (above 1.4), while, on the other hand, the varieties exhibiting the

297

lowest AX5:AX6 ratios (below 0.5) are all barley. This trend agrees with the results of

298

Dervilly-Pinel et al, 13 who found a higher proportion of di-substituted AX in barley than in

299

wheat. However, using our method, we were unable to clearly distinguish the wheat from the

300

barley varieties based on the BG3:BG4 ratio, although this ratio was previously reported to be

301

higher in wheat than in barley (3.0-4.5 and 1.8-3.5, respectively).21,

302

from our measurements were also lower than those documented. The mean value of

303

BG3:BG4 for the 10 varieties of wheat and 8 varieties of barley was 1.2 and 1.3, respectively,

304

with a standard deviation of 21%). Without excluding the possibility of bias in the estimation

305

of one of the BGOS species by the MALDI-MSI method, it can be assumed that any

306

misevaluation will evenly affect the BG3:BG4 ratio for all species and that this ratio could

307

still be used as a relative parameter for comparing the varieties.

308

For the eight barley cultivars (Figure 3) and for nine of the ten wheat cultivars (Figure 4),

309

some of the cross-sections were arbitrarily chosen from the central region of the grain and the

310

distribution of their AXOS and BGOS components were imaged by high-resolution MALDI-

311

MSI (i.e., at 50 µm spatial resolution). The AX5:AX6 and BG3:BG4 ratios were plotted with

312

a rainbow-color scale, where the color gradient from black to white reflects an increasing

313

value of AX5:AX6 and BG3:BG4 ratios. Some optical images of the same tissues were

314

acquired by fluorescence microscopy, thereby enabling colored pixels to be assigned to

315

specific cell structures of the endosperm. The average mass spectra obtained from selected

316

regions of some of the imaged sections are provided in Figure 5 and Figure 6. As shown in

317

Figure 3, the specific distribution of BG3 and BG4 species in the endosperm of the barley

318

grains was not obvious in any cultivar. Some of the cultivars (e.g., HGB4 and HGB7) actually

319

exhibited differently colored zones due to a gradient of increasing BG3 in these zones.

320

However, they do not correspond to regions of the endosperm with any known function, e.g.,

22

The values derived

14 ACS Paragon Plus Environment

Page 15 of 32

Journal of Agricultural and Food Chemistry

321

transfer cells or aleurone layer. Integrating the signal intensity of all released BG

322

oligosaccharides did not indicate a significant difference in the total amount of BG throughout

323

the endosperm in any of the cultivars (data not shown). This result is consistent with that of a

324

study by Zheng et al,40 who reported a uniform distribution of BG throughout the barley

325

grain.

326

In contrast to BG, pronounced differences in the substitution pattern of AX were observed

327

throughout the barley cross sections, as revealed by the monitoring of AX5:AX6. There were

328

also clear differences between the cultivars. In HGB2, a subtle gradient was observed from

329

the outer to the inner part of the endosperm. Mono-substituted AX (AX5) was more abundant

330

than di-substituted AX (AX6) in aleurone cells, while AX6 was more abundant in several

331

layers of the immediately adjacent cells, but AX5 was predominant in the vicinity of the

332

crease. Interestingly, an opposite gradient of AX5 and AX6 distribution was observed in

333

HGB7. HGB4 (and HGB3, although with less contrast) had slightly more AX5 in the aleurone

334

cells and markedly more AX5 in a small region close to the crease, while central cells had

335

more AX6 as indicated by black pixels, corresponding to an AX5:AX6 ratio below 1.2. An

336

inverse distribution was observed in HGB5, where the crease region was depleted in AX5,

337

AX5 was abundant in the aleurone layer, and central cells contained slightly more AX5 than

338

AX6. A similar pattern was reported by Wilson et al,

339

technique to identify highly substituted AX in the crease region of barley. HGB8 and HGB6

340

displayed a similar distribution pattern. The aleurone layer was enriched in AX5, while in the

341

rest of the tissue AX5 was moderately more abundant than AX6. Finally, HGB10 had uniform

342

distributions of AX5 and AX6 throughout the tissue, with slightly more AX5 than AX6.

343

In contrast to barley, molecular images of the BG distribution in wheat cultivars showed

344

smoother gradients and revealed some distinct areas: BG3 was prevalent in the central and

345

outer cells of all varieties, while BG4 was more abundant around the crease area (shown as

41

who used an antibody labelling

15 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 32

346

blue pixels in Figure 4). It was known that BG polymers were uniformly distributed

347

throughout the wheat endosperm cross sections.14,

348

variations of BG across the grain was unexplored until now. Our results reveal a

349

heterogeneous distribution of BG3 and BG4 units throughout the endosperm, with the transfer

350

cells having lower BG3:BG4 ratios. High ratios of BG3:BG4 were correlated to poor

351

solubility of BG polymers. 19 Thus, the low BG3:BG4 ratio in the crease region could indicate

352

a greater affinity of BG, and hence the cell walls, to water in accordance with the role of the

353

crease region in water transportation.

354

Molecular images of AX distribution in wheat revealed common features in the nine wheat

355

cultivars as follows: AX5 was predominant in the aleurone layer and immediately adjacent

356

cell layers, while the inner layers and the transfer cells region were enriched in AX6, as

357

previously reported for the 'Recital' cultivar. 14 However, the AX5:AX6 gradient had different

358

cultivar-specific patterns. It was more pronounced in the 'Magdalena' variety, while it was

359

smooth in the 'Thesee', 'Baltimore', 'Sisley', 'Tamaro' and 'Crousty' cultivars. On the other

360

hand, two cultivars: 'Malaca' and especially 'Aligre', exhibited very low AX5:AX6 throughout

361

the whole endosperm. These different patterns were in agreement with the structural

362

variations of AX observed using enzymatic fingerprinting. 3 Cultivars 'Aligre' and 'Malaca'

363

were clustered in one group, characterized by a high proportion of di-substitution, while

364

'Magdalena' and 'Virtuose' were clustered in a different group with highly mono-substituted

365

AX. Enzymatic fingerprinting revealed intermediate structural features for the other cultivars.

366

As mentioned earlier, the degree of substitution of AX was correlated to the water binding

367

ability of the polymer. 12 A higher A:X ratio was observed in the transfer cells of wheat grains

368

compared to the aleurone cells by Raman spectroscopy, 30 which was suggested to promote

369

the diffusion of water and nutrients by the transfer cells in the crease region. A deeper

370

investigation of the AX structural variation across the wheat endosperm was performed by

29

Yet, to our knowledge, the structural

16 ACS Paragon Plus Environment

Page 17 of 32

Journal of Agricultural and Food Chemistry

371

analyzing 17 consecutive sections, longitudinally cut from a grain of the 'Recital' cultivar. The

372

distribution profile had a “U” shape, representing the AX5/AX6 gradient from one lateral

373

side, through the crease and to the opposite lateral side of the wheat cross section (Figure 7).

374

The observed variations are fully consistent with the results of single cross sections from the

375

center of the grain, showing enrichment of mono-substituted AX at the periphery of cross

376

sections close to the pericarp and relative depletion in the crease.

377

In conclusion, this study is the first to provide a complete view of the selective distribution of

378

key structural features in AX and BG polymers across the endosperm of several wheat and

379

barley varieties. Cell walls play a major role in cellular compartmentalization, and MALDI-

380

MSI revealed that cultivars have strikingly different AX distributions within the endosperm

381

cell walls. These variations may influence local environments, impacting polymer assembly

382

and final grain properties. By providing new insights into the structural variability of cell wall

383

polysaccharides among cereals, MSI could thus advantageously be part of a strategy aiming at

384

understanding the structure-function relationships of these polymers.

385

Abbreviations used

386

AX-arabinoxylans; AXOS-arabinoxylan oligosaccharide; BG-beta glucans; BGOS-beta

387

glucans oligosaccharide; DP- degree of polymerization; MALDI-MSI- matrix-assisted laser

388

desorption/ionization mass spectrometry imaging; mXyl3-mono-substituted xylosyl residue at

389

O-3 position; dXyl-di-substituted xylosyl residue; RH-relative humidity; XXXG- Xyloglucan

390

heptasaccharide; Xyl-xylose.

391

Acknowledgment

392

This work was supported in part through a post-doctoral fellowship (Dušan Veličković) from

393

INRA (Institut National de Recherche Agronomique, France) and AgreenSkills.

17 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

394

Supporting Information Available

395

This material is available free of charge via the Internet at http://pubs.acs.org.

Page 18 of 32

396

18 ACS Paragon Plus Environment

Page 19 of 32

Journal of Agricultural and Food Chemistry

References 397

1.

Front. Plant. Sci. 2014, 5,456.

398 399

Burton, R. A.; Fincher, G. B., Evolution and development of cell walls in cereal grains.

2.

Fincher, G. B.; Stone, B. A., Cell walls and their components in cereal grain technology.

400

In Advances in cereal science and technology, Pomeranz, Y., Ed. American Association

401

of Cereal Chemists: St Paul, MN, 1986; pp 207-295.

402

3.

Ordaz-Ortiz, J. J.; Devaux, M. F.; Saulnier, L., Classification of wheat varieties based on

403

structural features of arabinoxylans as revealed by endoxylanase treatment of flour and

404

grain. J. Agric. Food Chem. 2005, 53, 8349-8356.

405

4.

cultivars from different locations of china. Food Chem. 2002, 79, 251-254.

406 407

Zhang, G. P.; Wang, J. M.; Chen, J. X., Analysis of beta-glucan content in barley

5.

Toole, G. A.; Le Gall, G.; Colquhoun, I. J.; Drea, S.; Opanowicz, M.; Bedo, Z.; Shewry,

408

P. R.; Mills, E. N. C., Spectroscopic analysis of diversity in the spatial distribution of

409

arabinoxylan structures in endosperm cell walls of cereal species in the HEALTGRAIN

410

diversity collection. J. Cereal Sci. 2012, 56, 134-141.

411

6.

Quraishi, U. M.; Murat, F.; Abrouk, M.; Pont, C.; Confolent, C.; Oury, F. X.; Ward, J.;

412

Boros, D.; Gebruers, K.; Delcour, J. A.; Courtin, C. M.; Bedo, Z.; Saulnier, L.; Guillon,

413

F.; Balzergue, S.; Shewry, P. R.; Feuillet, C.; Charmet, G.; Salse, J., Combined meta-

414

genomics analyses unravel candidate genes for the grain dietary fiber content in bread

415

wheat (Triticum aestivum L.). Funct. Integr. Genomic 2011, 11, 71-83.

416

7.

Shewry, P. R.; Piironen, V.; Lampi, A. M.; Edelmann, M.; Kariluoto, S.; Nurmi, T.;

417

Fernandez-Orozco, R.; Ravel, C.; Charmet, G.; Andersson, A. A. M.; Aman, P.; Boros,

418

D.; Gebruers, K.; Dornez, E.; Courtin, C. M.; Delcour, J. A.; Rakszegi, M.; Bedo, Z.;

419

Ward, J. L., The HEALTHGRAIN wheat diversity screen: Effects of genotype and

19 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 32

420

environment on phytochemicals and dietary fiber components. J. Agric Food Chem.

421

2010, 58, 9291-9298.

422

8.

Gebruers, K.; Dornez, E.; Boros, D.; Fras, A.; Dynkowska, W.; Bedo, Z.; Rakszegi, M.;

423

Delcour, J. A.; Courtin, C. M., Variation in the content of dietary fiber and components

424

thereof in wheats in the HEALTHGRAIN diversity screen. J. Agric. Food Chem. 2008,

425

56, 9740-9749.

426

9.

Nystrom, L.; Lampi, A. M.; Andersson, A. A. M.; Kamal-Eldin, A.; Gebruers, K.;

427

Courtin, C. M.; Delcour, J. A.; Li, L.; Ward, J. L.; Fras, A.; Boros, D.; Rakszegi, M.;

428

Bedo, Z.; Shewry, P. R.; Piironen, V., Phytochemicals and dietary fiber components in

429

rye varieties in the HEALTHGRAIN diversity screen. J. Agric. Food Chem 2008, 56,

430

9758-9766.

431

10. Andersson, A. A. M.; Lampi, A. M.; Nystrom, L.; Piironen, V.; Li, L.; Ward, J. L.;

432

Gebruers, K.; Courtin, C. M.; Delcour, J. A.; Boros, D.; Fras, A.; Dynkowska, W.;

433

Rakszegi, M.; Bedo, Z.; Shewry, P. R.; Aman, P., Phytochemical and dietary fiber

434

components in barley varieties in the HEALTHGRAIN diversity screen. J. Agric. Food

435

Chem. 2008, 56, 9767-9776.

436

11. Shewry, P. R.; Piironen, V.; Lampi, A. M.; Nystrom, L.; Li, L.; Rakszegi, M.; Fras, A.;

437

Boros, D.; Gebruers, K.; Courtin, C. M.; Delcour, J. A.; Andersson, A. A. M.; Dimberg,

438

L.; Bedo, Z.; Ward, J. L., Phytochemical and fiber components in oat varieties in the

439

HEALTHGRAIN diversity screen. J. Agric. Food Chem. 2008, 56, 9777-9784.

440 441

12. Saulnier, L.; Guillon, F.; Chateigner-Boutin, A. L., Cell wall deposition and metabolism in wheat grain. J. Cereal Sci. 2012, 56, 91-108.

442

13. Dervilly-Pinel, G.; Rimsten, L.; Saulnier, L.; Andersson, R.; Aman, P., Water-extractable

443

arabinoxylan from pearled flours of wheat, barley, rye and triticale. Evidence for the

20 ACS Paragon Plus Environment

Page 21 of 32

Journal of Agricultural and Food Chemistry

444

presence of ferulic acid dimers and their involvement in gel formation. J. Cereal. Sci.

445

2001, 34, 207-214.

446

14. Velickovic, D.; Ropartz, D.; Guillon, F.; Saulnier, L.; Rogniaux, H., New insights into

447

the structural and spatial variability of cell-wall polysaccharides during wheat grain

448

development, as revealed through MALDI mass spectrometry imaging. J. Exp. Bot. 2014,

449

65, 2079-2091.

450

15. Philippe, S.; Barron, C.; Robert, P.; Devaux, M. F.; Saulnier, L.; Guillon, F.,

451

Characterization using raman microspectroscopy of arabinoxylans in the walls of

452

different cell types during the development of wheat endosperm. J. Agric. Food Chem.

453

2006, 54, 5113-5119.

454

16. Toole, G. A.; Le Gall, G.; Colquhoun, I. J.; Nemeth, C.; Saulnier, L.; Lovegrove, A.;

455

Pellny, T.; Wilkinson, M. D.; Freeman, J.; Mitchell, R. A. C.; Mills, E. N. C.; Shewry, P.

456

R., Temporal and spatial changes in cell wall composition in developing grains of wheat

457

cv. Hereward. Planta 2010, 232, 677-689.

458

17. Ying, R. F.; Rondeau-Mouro, C.; Barron, C.; Mabille, F.; Perronnet, A.; Saulnier, L.,

459

Hydration and mechanical properties of arabinoxylans and beta-d-glucans films.

460

Carbohyd. Polym. 2013, 96, 31-38.

461

18. Shelat, K.; Vilaplana, F.; Nicholson, T.; How Wong, K.; Gidley, M.; Gilbert, R.,

462

Diffusion and viscosity in arabinoxylan solutions: Implications for nutrition. Carbohyd.

463

Polym. 2010, 82, 46-53.

464

19. Izydorczyk, M. S.; Dexter, J. E., Barley beta-glucans and arabinoxylans: Molecular

465

structure, physicochemical properties, and uses in food products-a review. Food Res. Int.

466

2008, 41, 850-868.

21 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 22 of 32

467

20. Shelat, K.; Vilaplana, F.; Nicholson, T.; Gidley, M.; Gilbert, R., Diffusion and rheology

468

characteristics of barley mixed linkage beta-glucan and possible implications for

469

digestion. Carbohyd. Polym. 2011, 86, 1732-1738.

470

21. Cui, W.; Wood, P. J.; Blackwell, B.; Nikiforuk, J., Physicochemical properties and

471

structural characterization by two-dimensional NMR spectroscopy of wheat beta-d-

472

glucan - comparison with other cereal beta-d-glucans. Carbohyd. Polym. 2000, 41, 249-

473

258.

474

22. Lazaridou, A.; Biliaderis, C. G., Molecular aspects of cereal beta-glucan functionality:

475

Physical properties, technological applications and physiological effects. J. Cereal. Sci.

476

2007, 46, 101-118.

477

23. Toole, G. A.; Le Gall, G.; Colquhoun, I. J.; Johnson, P.; Bedo, Z.; Saulnier, L.; Shewry,

478

P. R.; Mills, E. N. C., Spectroscopic analysis of diversity of arabinoxylan structures in

479

endosperm cell walls of wheat cultivars (Triticum aestivum) in the HEALTHGRAIN

480

diversity collection. J. Agric. Food Chem. 2011, 59, 7075-7082.

481 482

24. Burton, R. A.; Fincher, G. B., Current challenges in cell wall biology in the cereals and grasses. Front. Plant Sci. 2012, 3.

483

25. Guillon, F.; Tranquet, O.; Quillien, L.; Utille, J. P.; Ortiz, J. J. O.; Saulnier, L.,

484

Generation of polyclonal and monoclonal antibodies against arabinoxylans and their use

485

for immunocytochemical location of arabinoxylans in cell walls of endosperm of wheat. J

486

Cereal. Sci. 2004, 40, 167-182.

487

26. Philippe, S.; Tranquet, O.; Utille, J. P.; Saulnier, L.; Guillon, F., Investigation of ferulate

488

deposition in endosperm cell walls of mature and developing wheat grains by using a

489

polyclonal antibody. Planta 2007, 225, 1287-1299.

490 491

27. McCartney, L.; Marcus, S. E.; Knox, J. P., Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J. Histochem. Cytochem. 2005, 53, 543-546.

22 ACS Paragon Plus Environment

Page 23 of 32

Journal of Agricultural and Food Chemistry

492

28. Robert, P.; Jamme, F.; Barron, C.; Bouchet, B.; Saulnier, L.; Dumas, P.; Guillon, F.,

493

Change in wall composition of transfer and aleurone cells during wheat grain

494

development. Planta 2011, 233, 393-406.

495

29. Philippe, S.; Saulnier, L.; Guillon, F., Arabinoxylan and (1 -> 3),(1 -> 4)-beta-glucan

496

deposition in cell walls during wheat endosperm development. Planta 2006, 224, 449-

497

461.

498

30. Gessel, M. M.; Norris, J. L.; Caprioli, R. M., MALDI imaging mass spectrometry: Spatial

499

molecular analysis to enable a new age of discovery. J. Proteomics 2014, 107, 71-82.

500

31. Koeniger, S. L.; Talaty, N.; Luo, Y. P.; Ready, D.; Voorbach, M.; Seifert, T.; Cepa, S.;

501

Fagerland, J. A.; Bouska, J.; Buck, W.; Johnson, R. W.; Spanton, S., A quantitation

502

method for mass spectrometry imaging. Rapid Commun. Mass Sp. 2011, 25, 503-510.

503

32. Hankin, J. A.; Murphy, R. C., Relationship between MALDI IMS intensity and measured

504

quantity of selected phospholipids in rat brain sections. Anal Chem 2010, 82, 8476-8484.

505

33. Pirman, D. A.; Reich, R. F.; Kiss, A.; Heeren, R. M. A.; Yost, R. A., Quantitative

506

MALDI tandem mass spectrometric imaging of cocaine from brain tissue with a

507

deuterated internal standard. Anal. Chem. 2013, 85, 1081-1089.

508

34. Reich, R. F.; Cudzilo, K.; Levisky, J. A.; Yost, R. A., Quantitative MALDI-MSn analysis

509

of cocaine in the autopsied brain of a human cocaine user employing a wide isolation

510

window and internal standards. J. Am. Soc. Mass. Spectr. 2010, 21, 564-571.

511 512

35. Veličković, D.; Rogniaux, H., In situ digestion of wheat cell-wall polysaccharides. Bioprotocol 2014 4, e1306.

513

36. Ropartz, D.; Bodet, P. E.; Przybylski, C.; Gonnet, F.; Daniel, R.; Fer, M.; Helbert, W.;

514

Bertrand, D.; Rogniaux, H., Performance evaluation on a wide set of matrix-assisted laser

515

desorption ionization matrices for the detection of oligosaccharides in a high-throughput

23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 24 of 32

516

mass spectrometric screening of carbohydrate depolymerizing enzymes. Rapid Commun.

517

Mass Sp. 2011, 25, 2059-2070.

518

37. Nabipour, M.; Meskarbashee, M.; Farzad, S., Sodium and potassium accummulation in

519

different parts of wheat under salinity levels. Asian Journal of Agricultural Research

520

2007, 1, 97-104.

521 522

38. Al-Karaki, G. N., Germination, sodium, and potassium concentrations of barley seeds as influenced by salinity. J Plant Nutr. 2001, 24, 511-522.

523

39. Faure, R.; Courtin, C. M.; Delcour, J. A.; Dumon, C.; Faulds, C. B.; Fincher, G. B.; Fort,

524

S.; Fry, S. C.; Halila, S.; Kabel, M. A.; Pouvreau, L.; Quemener, B.; Rivet, A.; Saulnier,

525

L.; Schols, H. A.; Driguez, H.; O'Donohue, M. J., A brief and informationally rich

526

naming system for oligosaccharide motifs of heteroxylans found in plant cell walls. Aust.

527

J. Chem. 2009, 62, 533-537.

528 529

40. Zheng, G. H.; Rossnagel, B. G.; Tyler, R. T.; Bhatty, R. S., Distribution of beta-glucan in the grain of hull-less barley. Cereal Chem. 2000, 77, 140-144.

530

41. Wilson, S. M.; Burton, R. A.; Collins, H. M.; Doblin, M. S.; Pettolino, F. A.; Shirley, N.;

531

Fincher, G. B.; Bacic, A., Pattern of deposition of cell wall polysaccharides and transcript

532

abundance of related cell wall synthesis genes during differentiation in barley endosperm.

533

Plant Physiol. 2012, 159, 655-670.

534 535

24 ACS Paragon Plus Environment

Page 25 of 32

Journal of Agricultural and Food Chemistry

536

Figure captions

537

Figure 1. Schematized structure of (A) AX and (B) BG

538

Figure 2. Distribution of barley (red) and wheat (blue) cultivars according to the AX5:AX6

539

and BG3:BG4 ratios, as derived from the MALDI-MSI experiments. The number of sections

540

measured for determining AX5:AX6 and BG3:BG4 are given in parentheses, in that order.

541

Error bars correspond to the standard deviation of the mean observed in these experiments

542

(27%).

543

Figure 3. MALDI MS images of barley cultivars, plotting pixels according to the BG3:BG4

544

and AX5:AX6 ratios. Upper panels: fluorescence microscopy images of the same tissues. NB.

545

In the case of AX5:AX6 distributions, the blue pixels surrounding the tissue but outside of the

546

tissue are artefacts of the normalization procedure of the noise.

547

Figure 4. MALDI-MS images of wheat cultivars, plotting pixels according to the BG3:BG4

548

and AX5:AX6 ratios. Upper panels: fluorescence microscopy images of the same tissues.

549

Figure 5. MALDI-MSI spectra of BG in specific regions of barley seed.

550

Figure 6. Average MALDI-MSI spectra of AX in some wheat cultivars

551

Figure 7. Variability of released AX5 and AX6 in consecutive longitudinal cuts measured by

552

MSI. Error bars correspond to the technical variability of the experiment (20%). Images of the

553

AX5:AX6 ratio are depicted below for some characteristic longitudinal cuts (1st, 4th, 8th, 12th

554

and 17th sections).

555

25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 32

Figure graphics Figure 1

26 ACS Paragon Plus Environment

Page 27 of 32

Journal of Agricultural and Food Chemistry

Figure 2

4

Letter code for wheat varieties: Ali: Aligre Bal: Baltimore Cro: Crousty Mag: Magdalena Mal: Malacca Sis: Sisley Tam: Tamaro The: Thesee Vir: Virtuose Rec: Recital

3.5 Tam (2;3)

3

AX5:AX6

2.5

Rec (4;3)

2

The (3;2) Cro (3;4)

Vir (2;3)

1.5 Bal (4;3) Ali (2;3)

1

Mal (3;4) Sis (3;4)

0.5 HGB2 (4;3)

HGB10 (4;5)

Mag (3;3) HGB5 (3;3) HGB7 (4;3)

HGB6 (3;4)

HGB3 (3;4)

HGB8 (4;3)

HGB4 (2;5)

0 0.5

1

1.5

2

BG3:BG4

27 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 28 of 32

Figure 3

Figure 4

28 ACS Paragon Plus Environment

Page 29 of 32

Journal of Agricultural and Food Chemistry

Figure 5

29 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 32

Figure 6

556

557 558

30 ACS Paragon Plus Environment

Page 31 of 32

Journal of Agricultural and Food Chemistry

Figure 7

559 560

31 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 32 of 32

Table of Contents Graphics

561

32 ACS Paragon Plus Environment