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Characterization of Cell Wall Composition of Radish (Raphanus sativus L. var. sativus) and Maturation Related Changes Judith Schäfer, Anika Brett, Bernhard Trierweiler, and Mirko Bunzel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03693 • Publication Date (Web): 15 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Journal of Agricultural and Food Chemistry

Characterization of Cell Wall Composition of Radish (Raphanus sativus L. var. sativus) and Maturation Related Changes

Judith Schäfer†, Anika Brett†, Bernhard Trierweiler‡, Mirko Bunzel*,†

†

Institute of Applied Biosciences, Department of Food Chemistry and Phytochemistry,

Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131 Karlsruhe ‡

Max Rubner-Institut (MRI), Department of Safety and Quality of Fruit and Vegetables,

Haid-und-Neu-Str. 9, 76131 Karlsruhe

*Corresponding

author

(Tel:

+4972160842936;

Fax:

[email protected])

1 ACS Paragon Plus Environment

+4972160847255,

E-mail:


1

ABSTRACT

2

Cell wall composition affects the texture of plant-based foods. In addition, the main

3

components of plant cell walls are dietary fiber constituents and made responsible for

4

potential physiological effects that are largely affected by the structural composition of the

5

cell walls. Radish (Raphanus sativus L. var. sativus) is known to develop a woody and firm

6

texture during maturation and ripening, most likely due to changes in the cell wall

7

composition. To describe these changes chemically, radish was cultivated and harvested at

8

different time points, followed by detailed chemical analysis of insoluble fiber

9

polysaccharides and lignin. During maturation, changes in polysaccharide profiles were

10

observed, with a decrease in the portion of neutral pectic sidechains and an increase in the

11

xylan portion being predominant. Radish lignin was characterized by unexpectedly high

12

incorporation of p-coumaryl alcohol into the polymer. Maturation dependent increases in

13

lignin contents were accompanied by compositional changes of the lignin polymers with

14

sinapyl alcohol being preferentially incorporated.

15 16

KEYWORDS

17

Radish, maturation related changes, cell wall composition, cell wall polysaccharides, lignin

18

characterization, dietary fiber

19


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INTRODUCTION

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Plant cell walls mainly consist of polysaccharides such as cellulose, hemicelluloses, and

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pectins as well as structural proteins. In addition, lignin may be incorporated after completion

23

of cell wall growth. Thus, the composition of plant cell walls depends on the taxonomic

24

position of the plant as well as the tissue, the cell type, and maturity of the plant. For example,

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primary cell walls of dicotyledonous plants are mainly composed of cellulose, xyloglucans,

26

pectins, and structural proteins, whereas secondary cell walls contain higher amounts of

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cellulose, xylans, and lignin but less xyloglucans.1 Both pre- and post-harvest ripening of

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fruits including the resulting changes in the cell wall composition is comparatively well

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described in literature.2, 3 Fruit ripening is often accompanied by softening of the fruit caused

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by degradation of pectins in the middle lamella and the primary cell wall. Especially the

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degradation of arabinan and galactan structures is a characteristic of fruit ripening. Galactan

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degradation is often described during preharvest development, whereas degradation of

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arabinans occurs during storage of fruits.4,

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described during the early stage of preharvest ripening resulting in loss of network structures

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within the cell wall.6 In contrast, several vegetables develop firm and woody texture during

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maturation; however, changes in the cell wall composition of vegetables are not well

37

understood. Woody texture is often associated with both enhanced incorporation of lignin and

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formation of cell wall cross-links, which was confirmed for stored asparagus.7-11

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Changes in cell wall composition of plant-based foods potentially affect both texture and

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nutritional benefits. The main components of plant cell walls belong to the dietary fiber

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complex. In general, dietary fiber is known to be beneficial for health,12 depending on dietary

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fiber intake, dietary fiber composition, the detailed structures of individual dietary fiber

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components, and their covalent and non-covalent interactions. For example, lignified fibers

44

are suggested to reduce colon cancer risk by adsorbing carcinogens such as heterocyclic

5

In addition, degradation of xyloglucans is



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aromatic amines, which is affected by both lignin content and structure.13, 14 The composition

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and detailed structures of fiber polysaccharides determine the extent of fermentation and the

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pattern of fermentation products formed in the colon.12 Therefore, a detailed characterization

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of dietary fiber structures is required to evaluate potential fiber related health benefits of

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plant-based foods.

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To the best of our knowledge, detailed data describing the cell wall composition of radish

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(Raphanus sativus L. var. sativus) (also known as small or European radish) is rarely

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available. A few authors determined the monosaccharide

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polysaccharides from closely related radish (Raphanus sativus L.)15-18 or lignin contents and

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monolignol ratios.19, 20 However, a detailed characterization of the whole cell wall of radish

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(Raphanus sativus L. var. sativus) and its dependence on the stage of maturity has not been

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performed yet. Therefore, this study characterizes the composition of insoluble cell wall

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components of radish, including both polysaccharides and lignin. In addition, maturation

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related modifications of the characterized structures are described.

59 60


composition of fiber

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MATERIALS AND METHODS

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Chemicals

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Heat-stable α-amylase Termamyl 120 L (from Bacillus licheniformis, 120 KNU/g), the

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protease Alcalase 2.5 L (from Bacillus licheniformis, 2.5 AU/g), and the amyloglucosidase

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AMG 300 L (from Aspergillus niger, 300 AGU/g) were from Novozymes (Bagsvaerd,

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Denmark); endo-arabinanase (from Aspergillus niger, 200 U/mL) and endo-galactanase (from

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Aspergillus niger, 1300 U/mL) were from Megazyme (Bray, Ireland); the complex

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carbohydrase mixture Driselase (from Basidiomycetes) was from Sigma-Aldrich (St. Louis,

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MO). Chemicals used, including deuterated NMR solvents, were either from Sigma-Aldrich,

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Roth (Karlsruhe, Germany), VWR (Radnor, PA), or Alfa Aesar (Ward Hill, MA).

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Hexadeuterated p-hydroxycinnamyl alcohol diacetates were synthesized as previously

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described.20

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Plant material

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Radish (Raphanus sativus L. var. sativus) was grown from seed in a greenhouse in January

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2015 and cultivated for about 20 weeks under normal day/night changes. Temperature was

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not lower than 15 °C. Additional light was given for up to 10 h when necessary. The first

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batch (HA1) was harvested eight weeks after sowing. This time point represents the time

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point at which radishes are normally harvested. The following harvests were 3 (HA2), 6

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(HA3), 9.5 (HA4), and 11.5 (HA5) weeks after the first harvest. Samples were frozen and

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freeze-dried directly after harvest.

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Preparation of insoluble fiber

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The freeze-dried material was milled to a particle size of 70%).11 Nevertheless,

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the comparably low portions of β-aryl-ether did not dramatically affect the information about

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monolignol composition obtained from the DFRC method, which is (with exception of HA2)

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roughly comparable to the results of the 2D-NMR analysis. Because the DFRC method

339

selectively cleaves β-O-4-linkages to determine monolignols, only monomers that are

340

involved in these linkages are captured, whereas all monolignols of the isolated lignin

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polymers are determined by using 2D-NMR approach.

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Substantial changes in the monolignol composition were observed during maturation of radish

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(Table 3). The portion of S units (DFRC method) increased from 17.0% (HA1) to 46.1%

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(HA5, 11.5 weeks after HA1), resulting in a decrease of the G/S ratio from about 4 (HA1) to

345

1 (HA5). The increase of sinapyl alcohol used for lignin synthesis during maturation was also

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confirmed by 2D-NMR (Table 3). Sinapyl alcohol was preferentially incorporated into the

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lignin polymers by both β-O-4 linkages and β-1 linkages as shown by an increase of these

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linkage types (Table 4). Both linkage types are mainly formed by adding a new monolignol to

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existing lignin polymers, which represents the usual lignification process in its advanced 16 ACS Paragon Plus Environment

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state.37 The decreased portion of dibenzodioxocin structures among the interunit linkages with

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increased maturation can be explained by the increased incorporation of sinapyl alcohol,

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which is less involved in the formation of dibenzodioxocin structures due to the occupied

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carbon-5 position at the aromatic ring.

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HMBC spectra of the isolated and acetylated lignin polymers were analyzed to get

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information about the monolignols involved in the various linkage types. Identification of the

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linkage types through which p-coumaryl alcohol was incorporated into the polymer was of

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particular interest. Unfortunately, however, we were not able to link any of the linkage types

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to p-coumaryl alcohol under the conditions (lignin amounts, spectroscopic conditions) used if

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lignins isolated from HA1 and HA2 radishes were analyzed. HMBC experiments of lignins

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isolated from HA4 radishes (Figure 4) showed that both β-aryl-ethers and phenylcoumaran

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units result from coupling of coniferyl and sinapyl alcohol to the existing polymer.

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Dibenzodioxocin units result mainly from coniferyl alcohol. In contrast, spirodienone and

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resinol structures almost entirely derive from sinapyl alcohol coupling. Resinol units are

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generally found to be formed from monolignol dimerization, especially from sinapyl

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alcohol,37 whereas both coniferyl or sinapyl alcohol can be involved in the formation of

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spirodienone structures.40 In radish, spirodienone structures appear to result primarily from

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sinapyl alcohol coupling to the existing lignin polymers, which is in accordance with our

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finding that spirodienone structures were increased with increasing sinapyl alcohol

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incorporation during maturation. This is, however, in contrast to lignins from other plant-

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based foods such as kiwi or pear where spirodienone units result primarily from coniferyl

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alcohol.33

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In conclusion, distinct changes in the plant cell wall composition were analyzed during

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maturation of radishes. Some of these changes such as increased portions of xylans and lignin

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are most likely due to the deposition of secondary cell walls with maturity, whereas other 17 ACS Paragon Plus Environment


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structural changes such as altered pectin structures cannot be fully explained at this point. The

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changes described go along with textural changes of the radishes. During maturation the

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texture of radishes developed woody and chewy properties and got less juicy, tender and

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crisp. In addition, changes of the cell wall composition should also affect the physiological

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properties of the non-protein cell wall components as dietary fiber constituents.

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SUPPORTING INFORMATIONS

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Figure: Textural properties of radishes used in this study and their changes during maturation

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(harvest 1-4). Table: Monomer compositions of radish insoluble fiber polysaccharides

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determined after H2SO4-hydrolysis and methanolysis. The Supporting Information is available

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free of charge via the Internet at http://pubs.acs.org.

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ABBREVIATIONS USED

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ABSL, acetyl bromide soluble lignin; DFRC, derivatization followed by reductive cleavage;

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G, guaiacyl; H, p-hydroxyphenyl; HA, harvest; HPAEC-PAD, high-performance anion

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exchange chromatography with pulsed amperometric detection; PMAAs, partially methylated

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alditol acetates; S, sinapyl; SIM, selected ion monitoring.

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ACKNOWLEDGMENT

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This work was funded by a fellowship from Carl Zeiss foundation.

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FIGURE CAPTIONS Figure 1: Monomer composition (in mol%) of radish insoluble fiber polysaccharides. The monosaccharides were determined by high-performance anion exchange chromatography with pulsed amperometric detection after both H2SO4-hydrolysis (A) and methanolysis (B). Results (n=3) are shown from harvest one to five (HA1-HA5). Due to underestimation of uronic acids after H2SO4-hydrolysis they were not used to calculate monosaccharide ratios in A). For HA3-HA5, rhamnose (Rha) was identified but the values were below the tested concentration range. GalA, galacturonic acid; Man, mannose; Xyl, xylose; Glc, glucose; Gal, galactose; Ara, arabinose; Fuc, fucose. Figure 2: Structures of identified arabino- (A) and galacto-oligosaccharides (B) liberated from insoluble fiber polysaccharides of radish samples, which were harvested at different time points. The oligosaccharides were analyzed after incubation of the sample material with endoarabinanase and endo-galactanase, followed by high-performance anion exchange chromatography with pulsed amperometric detection analysis of the hydrolysates. Figure 3: HSQC spectrum (sidechain region) of acetylated lignins isolated from radish insoluble fiber (harvest four, HA4) measured in acetone-d6. Volume integration of the signals representing α-carbon-proton couplings results in linkage type profiles given in Table 3. Figure 4: Partial HMBC spectrum of acetylated lignins isolated from radish insoluble fiber (harvest four, HA4). The highlighted bands show correlations of α-protons (representing different linkage types) with carbons within three bonds. Horizontal bands indicate diagnostic couplings to carbons-2 and -6 of the aromatic rings of guaiacyl (G) and syringyl (S) units. In addition, identified correlations of the α-protons to β- and γ-carbons are indicated (if present). A, β-aryl-ether (β-O-4); B, phenylcoumaran (β-5); C, resinol (β-β); D, dibenzodioxocin (55/β-O-4); X1, cinnamyl alcohol; SD, spirodienone (β-1/α-O-α); F, traditional β-1. 23 ACS Paragon Plus Environment


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TABLES Table 1 Ratios (mol%) of Partially Methylated Alditol Acetates (PMAAs) Resulting From Methylation Analysis of Radish Insoluble Fiber (n=2). The Results are Shown for all Harvest Time Points (HA1-HA5). t, Terminal; p, Pyranose; f, Furanose; Glc, Glucose; Xyl, Xylose; Gal, Galactose; Ara, Arabinose; Rha, Rhamnose; Man, Mannose. PMAAs [mol%]

HA1

HA2

HA3

HA4

HA5

t-Glcp 1,4-Glcp 1,4,6-Glcp

1.36 ± 0.01 59.74 ± 1.38 4.47 ± 0.06

1.14 ± 0.05 62.04 ± 1.67 4.76 ± 0.49

1.92 ± 0.005 57.41 ± 3.92 4.87 ± 0.26

2.12 ± 0.01 58.36 ± 1.88 3.75 ± 0.20

2.25 ± 0.13 55.58 ± 2.49 2.89 ± 0.06

t-Xylp 1,4-Xylp

3.73 ± 0.03 4.68 ± 0.56

4.21 ± 0.23 5.05 ± 0.09

4.03 ± 0.04 8.54 ± 0.51

4.91 ± 0.61 14.39 ± 0.03

4.38 ± 0.08 19.72 ± 2.16

t-Galp 1,4-Galpa

3.20 ± 0.01 2.05 ± 0.006

3.00 ± 0.02 1.94 ± 0.11

2.35 ± 0.06

1.81 ± 0.19

1.51 ± 0.09

t-Araf 1,2-Araf 1,5-Araf 1,3,5-Arafb 1,2,5-Araf 1,2,3,5-Araf

5.78 ± 0.34 0.58 ± 0.03 6.60 ± 0.23 1.98 ± 0.03 0.87 ± 0.07 0.93 ± 0.07

5.13 ± 0.13 0.53 ± 0.03 4.41 ± 0.20 2.00 ± 0.70 0.61 ± 0.12 1.01 ± 0.12

4.99 ± 0.04 0.73 ± 0.12 6.83 ± 2.75 2.72 ± 0.18 0.49 ± 0.02 0.88 ± 0.23

3.81 ± 0.27 0.59 ± 0.26 3.02 ± 0.57 2.38 ± 0.02 0.35 ± 0.02 0.66 ± 0.11

3.35 ± 0.17 0.94 ± 0.07 2.87 ± 0.51 1.87 ± 0.09 0.20 ± 0.01 0.44 ± 0.02

1,2-Rhap 1,2,4-Rhap

0.56 ± 0.20 0.66 ± 0.05

0.54 ± 0.08 0.64 ± 0.12

1.08 ± 0.82 0.49 ± 0.01

1.64 ± 0.69 0.20 ± 0.05

1.88 ± 0.01 0.19 ± 0.02

1,4-Manp 1,4,6-Manp

2.19 ± 0.02 0.63 ± 0.11

2.40 ± 0.17 0.60 ± 0.08

1.98 ± 0.10 0.69 ± 0.05

1.58 ± 0.03 0.41 ± 0.03

1.61 ± 0.08 0.30 ± 0.001

a

GC-MS identification was not possible for HA3, HA4, and HA5. The presence of galactans

was confirmed by the determination of galacto-oligosaccharides. b

Overestimation possible due to incomplete peak separation during GC-FID analysis.


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Table 2 Lignin Contents of Radish Insoluble Fiber Isolated From Radish Samples Harvested at Different Time Points (HA1-HA5). Lignin Contents were Determined as Klason Lignin and Acetyl Bromide Soluble Lignin (ABSL) (n=3). Klason lignin

ABSL

[g/100 g insoluble fiber]

[g/100 g insoluble fiber]

HA 1

5.05 ± 0.96

1.09 ± 0.005

HA 2

5.29 ± 1.52

1.43 ± 0.12

HA 3

7.87 ± 0.27

2.82 ± 0.16

HA 4

14.98 ± 0.33

7.85 ± 0.32

HA 5

16.33 ± 0.31

9.56 ± 0.38



Table 3 Monomer Composition of Lignin Polymers Isolated from Radish Harvested at Different Time Points (HA1-HA5). The Monomer Compositions of the Lignin Polymers Result from the Derivatization Followed by Reductive Cleavage (DFRC) Method (n=3) and 2D-NMR Analysis (n=1). H, p-Hydroxyphenyl; G, Guaiacyl; S, Syringyl.

H:G:S (DFRC)

H:G:S (NMR)

HA1

14.0 : 69.0 : 17.0

20.7 : 58.0 : 21.2

HA2

10.6 : 76.8 : 12.6

10.8 : 66.8 : 22.4

HA3

4.7 : 60.5 : 34.7

2.1 : 68.7 : 29.3

HA4

3.0 : 52.5 : 44.5

-a : 62.1 : 37.9

HA5

2.3 : 51.6 : 46.1

-a : 55.3 : 44.7

a

Identification not possible due to overlapped, unknown signals.


Page 26 of 34

Page 27 of 34


Table 4 Linkage Type Profiles of Lignin Polymers Isolated from Radish Harvested at Different Time Points (HA1-HA5). The Linkage Type Distributions were Determined by Volume Integration of the Signals Resulting from the Respective α-Carbon-Proton Correlations in the HSQC Spectra (n=1). Integral Correction Factors Described in Literature

34

were Used for the

Determination of β-Aryl-Ether (β-O-4, A), Phenylcoumaran (β-5, B), Resinol (β-β, C), and Dibenzodioxocin (5-5/β-O-4, D) Portions. For Cinnamyl Alcohol (X1), Spirodienone (β-1/αO-α, SD), and Traditional β-1 Linkages (F) an Integral Correction Factor of 1 was Assumed.

%A

%B

%C

%D

%X1

%SD

%F

HA1

56.7

7.9

19.0

6.5

1.4

2.6

6.0

HA2

64.0

7.2

14.6

6.4

1.2

4.0

2.5

HA3

59.6

7.6

17.6

4.1

3.1

2.0

6.0

HA4

61.3

6.7

15.8

2.1

3.4

2.9

7.8

HA5

58.8

6.5

15.9

1.7

3.6

4.4

9.0



FIGURE GRAPHICS Figure 1


Page 28 of 34

Page 29 of 34


Figure 2

A-2a

A)

A-3a

A-4b

O

5

O

O OH

OH

OH

O

O

OH OH

1

OH

HO OH

OH

1

O

OH

1

1

HO

OH

OH OH

HO

O OH

OH O

O O

OH

HO

O

O

5 3

O OH O

O

O 2 O

OH

O

OH

5 3

O

OH

OH

OH

O

OH

O

O

O

O OH

OH

B) G-4b

G-2a

O HO

1 O OH OH 6

HO OH O

O 1

HO

O OH

OH

OH 4

1

HO

O OH

4

O

OH OH

OH 4

O

HO

O OH

1

HO

O OH

HO

1

5 3

1

HO

OH

OH

HO OH

O

O O

OH

O

OH


OH

5 3

O

O

1

OH

1

1

HO

OH

HO

OH

5 OH

5

O

O

OH

OH HO

O

OH

5

1

5 3

O

O

1

HO

O

O

1

O OH

O OH

OH

OH

HO OH

OH

5

2

OH

A-7b

OH

HO

OH

O

OH

A-7a

OH

OH

1

OH

A-5a

O

5 3

O

O

OH 1

O

HO

HO

O

O OH

HO

OH

5

O

O

5

O

O

OH

O OH

5

2

5

O

O

OH

OH

OH

5

O

O

OH

1

HO

OH

OH

A-4a

O

O OH

OH

OH 1

OH


Figure 3


Page 30 of 34

Page 31 of 34


Figure 4



TABLE OF CONTENTS GRAPHIC

polysaccharide and lignin characterization


Page 32 of 34

Aγ Aα Fα

Sα Dα

Cγ Cα

Cγ

HO

α

γ β

Dβ

OMe

γ

α

O

B phenylcoumaran (β-5)

5 5 MeO

O

120

β

X1α 4

F2 [ppm]

ACS Paragon Plus Environment

HO

O

α γ β 1

OH

SD spirodienone (β-1/α-O-α)

β γ

OMe OH

γ β

1

methoxy OMe

OR OMe

O

α

O O

α

D dibenzodioxocin (5-5/β-Ο−4)

C resinol (β-β) HO

OH

X1 cinnamyl alcohol 5

γ β

O

γ β α

HO

α

6

5

HO

A β-aryl-ether (β-O-4)

100

Bα

Aβ

Bγ 80

X1γ

Sβ

HO

60

Cβ

Bβ

F1 [ppm]


O

Page 33 of 34

OH

F traditional β-1 linkage

unresolved unassigned, saccharides etc.


SDα

Bα

β-O-4

β-1

β-5

Dα

5-5/ β-O-4

β

β-β

β β

γ

60

γ

Cα F1 [ppm]

Aα

Page 34 of 34

γ

100

80

β

S2/6 G2

120

G6 6.0

5.8

5.6

5.4

ACS Paragon Plus Environment

5.2

5.0

4.8

F2 [ppm]

Raphanus sativus L. var. sativus - ACS Publications - American

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