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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

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Journal of Agricultural and Food Chemistry

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

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

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Polysaccharides According to Their Main Structural Features

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

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Hélène Rogniaux *

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

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

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

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1 ACS Paragon Plus Environment


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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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function relationships of these two polymers.

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

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wall, MALDI-MS imaging, plant, polysaccharides.


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Introduction

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

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

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

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

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

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

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

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

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

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variability is likely driven by genetics,6,

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

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

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

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

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

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

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1.4-2.2% dm for barley.8-11

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

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

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

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

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

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

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

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and its biological significance is far from



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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AX.12,

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

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the grain.18

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

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

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

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

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

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

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

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


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

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

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

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

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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

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for rye and 1.5-2.3 for oats.21,

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

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

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

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

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

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

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

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

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structural changes in these polymers.5,

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

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

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

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

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

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

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

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

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

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

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Although little information is currently available on the

15, 28, 29

Recently, our group proposed an imaging



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

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

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

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

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DP4 released from the BG.

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Materials and Methods

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Wheat samples

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

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

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

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

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

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

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

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

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Barley samples

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

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

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

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

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

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

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ranged from 3.7 up to 6.5% dm.10

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Chemicals and reagents 6 ACS Paragon Plus Environment

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

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

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

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

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

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

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

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

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

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

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France).

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Enzymes

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

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

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

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

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

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

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

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

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

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Sample preparation for MALDI-MS imaging: removal of starch

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

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



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

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

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

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

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a support.

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In situ digestion of cell wall polysaccharides

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

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

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

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

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

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

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

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enzymatic digestion of ground cell wall material.35

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MALDI matrix

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

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oligosaccharides

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potassium adducts caused by the high concentrations of potassium in wheat

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

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Application of the MALDI matrix for MSI

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

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

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

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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


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

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

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MSI Analysis

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

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

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

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

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

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

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

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

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

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

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

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

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Daltonics) software package.

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Quantification of released oligosaccharides and their variation between samples

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

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corresponding to the enzymatically released oligosaccharide AX5 (XA3XX according to

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

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

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

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

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



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

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

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

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

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

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

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Microscopy

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

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

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

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

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

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

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

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

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

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

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

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aleurone layer and the pericarp.

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Results and Discussion

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

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

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

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


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

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

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

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

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

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properties. 18,

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

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

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

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

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

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Qualification of the method

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

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

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varieties by MALDI-MSI.

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

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

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

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

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

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

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

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

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

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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



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

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

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

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

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20% in the rest of the study.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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distribution in the grains

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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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


Page 15 of 32


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



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


Page 17 of 32


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.



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


Page 19 of 32


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

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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.

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534 535


Page 25 of 32


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



Page 26 of 32

Figure graphics Figure 1


Page 27 of 32


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



Page 28 of 32

Figure 3

Figure 4


Page 29 of 32


Figure 5



Page 30 of 32

Figure 6

556

557 558


Page 31 of 32


Figure 7

559 560



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Table of Contents Graphics

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Cultivars - ACS Publications - American Chemical Society

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