Research article

Post-translational processing of

-xylanases and changes in

extractability of arabinoxylans during wheat germination

Evelien De Backer

, Kurt Gebruers

, Wim Van den Ende

, Christophe M. Courtin

, Jan A. Delcour

Laboratory of Food Chemistry and Biochemistry, and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven,

Kasteelpark Arenberg 20 bus 2463, B-3001 Leuven, Belgium

Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, bus 2434, B-3001 Leuven, Belgium

a r t i c l e i n f o

Article history:
Received 1 July 2009
Accepted 31 October 2009
Available online 6 November 2009

Keywords:
Wheat
Endogenous

-xylanase

Arabinoxylan

a b s t r a c t

Endo-1,4-

-xylanase (EC 3.2.1.8,

-xylanase) activity, and arabinoxylan (AX) level and extractability

were monitored for the

ﬁrst time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to

24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly
three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in
a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3

e4 DPI). This

increase was not accompanied by the discernible presence of homologues of the barley X-I

-xylanase

as established by immunodetection. This suggests that other, yet unidenti

ﬁed

-xylanases operate in

this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of
unprocessed 67 kDa X-I like

-xylanase, which was much more abundant in the presence of GA. The

occurrence of higher levels of the unprocessed enzyme was related with higher

-xylanase activities

and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa

-xylanase.

During the late stage (up to 24 DPI) gradual processing of the 67 kDa

-xylanase occurred and was

associated with a drastic increase in

-xylanase activity. Up to 120-fold higher activity was recorded at

24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during
the late stage, suggesting that the

-xylanase is processed into more active forms to achieve extensive

AX breakdown.

1. Introduction

Arabinoxylans (AXs) are one of the most important non-starch

polysaccharides in cereal cell walls

[32]

. Wheat endosperm AX

consists of a linear backbone of

-(1,4)-linked

-xylopyranosyl

residues, which can be substituted with

-arabinofuranosyl resi-

dues at the C(O)-2 and/or C(O)-3 position. Phenolic acids such as
ferulic acid and coumaric acid can be esteri

ﬁed to the C(O)-5 position

of the arabinose substituents

[8,24]

. In wheat bran, the main AXs are

glucuronoarabinoxylans. Typical for glucuronoarabinoxylans is the
presence of uronic acids, mostly (4-O-methyl-)glucuronic acid, at
the C(O)-2 position of some xylopyranosyl residues

[36]

Malting experiments with barley have shown that the total AX

level decreases

[30,25]

while the water extractable AX (WEAX) level

increases during germination (4

e7 days)

[30,40]

. For wheat, Fincher

and Stone

[19]

described that during a four day incubation period of

embryo-free wheat grains in the presence of gibberellic acid (GA) the
level of WEAX increased threefold. During barley germination, AXs
of decreasing degree of substitution are extracted

[40]

. In developing

barley coleoptiles, the total AX content as a percentage of the total
cell wall material slightly increases, while the arabinose to xylose
(A/X) ratio decreases

[23]

. It has been suggested that the arabinose

substituents are progressively removed during the growth of the
coleoptile to allow deposition of AX in the cell wall

[22]

Endo-1,4-

-xylanases (EC 3.2.1.8,

-xylanases) are key

enzymes in the breakdown of AX

[14]

. They hydrolyse the xylan

backbone of AX internally and cause drastic changes in the physi-
cochemical properties of the polysaccharide. Based on sequence
homology and hydrophobic cluster analysis,

-xylanases are

classi

ﬁed into different glycoside hydrolase (GH) families

[27]

. Most

of them known today are from microbial origin and belong to GH
families 10 and 11

[27,14]

. Much less is known of their counterparts

in plants, which are exclusively classi

ﬁed in GH family 10.

Abbreviations: AX, arabinoxylan; AXs, arabinoxylans; A/X ratio, arabinose to

xylose ratio; BSA, bovine serum albumin; DPI, days post-imbibition; GA, gibberellic
acid; GH, glycoside hydrolase; mXU, milli-

-xylanase unit; PAbs, polyclonal

antibodies; PCD, programmed cell death; TAXI, Triticum aestivum

-xylanase

inhibitor; WEAX, water extractable arabinoxylan; WUAX, water unextractable
arabinoxylan; XIP,

-xylanase inhibiting protein.

* Corresponding author. Tel.: þ32 16321919; fax: þ32 16321997.

E-mail address:

kurt.gebruers@biw.kuleuven.be

(K. Gebruers).

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Plant Physiology and Biochemistry 48 (2010) 90

e97

It is widely accepted that

-xylanases, in concert with other

cell wall degrading enzymes such as 1,3-1,4-

-glucanases, are

involved in the progressive degradation of aleurone and endo-
sperm cell walls during grain germination. Cell wall loosening and
disassembly facilitate the mobilisation of storage proteins and
polysaccharides for the developing embryo

[7,3,39]

. The production

of hydrolytic enzymes is induced by GA, a plant hormone produced
by the embryo of germinating grains

[4,35,1]

. Exogenous applica-

tion of GA promotes germination

[28]

and increases

-xylanase

activity during germination of barley

[11]

In cereal grains, barley X-I

-xylanase (GenBank accession

numbers AF287726 and AJ849364) is the best characterized

xylanase. Active forms of 41 kDa and 34 kDa, both proteolytically
derived from the 61.5 kDa X-I

-xylanase precursor, have been

described by Caspers et al.

[7]

. In contrast to many other hydrolytic

enzymes, the barley X-I enzyme does not carry a signal peptide for
extracellular secretion after translation. It is, hence, a cytosolic
enzyme accumulating in e.g. aleurone cells that is only released after
programmed cell death (PCD). Proteolytic processing of the X-I
enzyme might be essential to increase its activity and mobility

[7]

Caspers et al.

[7]

concluded that the major function of barley X-I

xylanase is the degradation of aleurone and endosperm cell walls
during the later stages of barley germination. The X-I enzyme is one
of two isozymes in barley described later on by Van Campenhout and
Volckaert

[42]

. These authors monitored the presence of mRNA of

two isozymes, i.e. the X-I and X-II

-xylanases (GenBank accession

numbers AJ849365 and AJ849366), during germination and plant
and ear development, and demonstrated that these two isozymes
differ in spatial and temporal expression. During germination, the
X-I enzyme is synthesized in the aleurone and scutellum, while
the X-II enzyme is synthesized in the roots, shoot and scutellum. The
former is presumed to assist in the release of nutrients for the rapidly
developing embryo, while the latter has been hypothesized to be
involved in the cell wall metabolism of e.g. vascular tissues

[42]

. So

far, the amino acid sequence of one wheat

-xylanase (AF156977)

has been reported

[37]

. The wheat enzyme is highly homologous to

the barley X-I (88% identity, 93% similarity) and X-II (81% identity,
87% similarity)

-xylanases

[38]

While many microbial

-xylanases are strongly inhibited

by cereal proteinaceous

-xylanase inhibitors, so far, sensitivity

of plant

-xylanases towards the well-studied Triticum aestivum

-xylanase inhibitors (TAXI)

[13,20]

-xylanase inhibiting

proteins (XIP)

[33,21]

or thaumatin-like

-xylanase inhibitors

[18]

has not yet been reported.

Earlier reports in the

ﬁeld of this paper dealt either with the

changes in AX level and solubility or with the changes in

xylanase activity and enzyme processing during germination and
seedling growth, with an emphasis on barley. Only Sungurtas and
co-workers

[40]

studied

-xylanase activity and AX solubilisation

together in barley during a short malting process (7 days). We here
describe the

ﬁrst comprehensive study on changes in wheat

xylanase activity, and AX level and extractability during wheat
germination and subsequent seedling growth, up to 24 days post-
imbibition (DPI). Immunoblotting allowed monitoring the presence
of wheat

-xylanase as well as the forms derived from it by

proteolytic processing.

2. Materials and methods

2.1. Materials

Wheat cultivar Glasgow was obtained from Clovis Matton

(Avelgem, Belgium). Recombinant barley X-I

-xylanase (GenBank

accession number AF287726) was the one described by Van
Campenhout et al.

[41]

. Polyclonal antibodies (PAbs) against this

His-tagged recombinant barley

-xylanase

[41]

were produced by

Eurogentec (Luik, Belgium) using the

“87-day classic polyclonal

antibody protocol

”. TAXI and XIP were isolated from wheat by the

ﬁnity-based puriﬁcation procedure described by Gebruers et al.

[21]

. Azurine cross-linked wheat AX tablets (Xylazyme-AX tablets)

were from Megazyme (Bray, Ireland). Electrophoretic media were
from Bio-Rad Laboratories (Hercules, CA, USA). The low molecular
mass markers (GE Healthcare, Uppsala, Sweden) used for SDS-PAGE
were phosphorylase b (94.0 kDa), bovine serum albumin (BSA,
67.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (30.0 kDa),
soybean trypsin inhibitor (20.1 kDa) and

-lactalbumin (14.4 kDa).

Protran nitrocellulose transfer membranes (pore size 0.45

(Whatman GmbH, Dassel, Germany) were used for immunoblotting.
All other chemicals and reagents, BSA and horseradish peroxidase
conjugated goat antibodies against rabbit IgG were purchased from
Sigma

eAldrich (Bornem, Belgium) and were of analytical grade

unless speci

ﬁed otherwise.

2.2. Seed germination and sampling

Wheat kernels were germinated in a pilot-scale micromalting

system (Joe White Malting Systems, Perth, Australia) under optimal
conditions of temperature, humidity and aeration for imbibition
and germination. The micromalting system was sterilized using
house hold bleach [3.0% (w/v) active chlorine] prior to use. Imbi-
bition was performed by submerging the kernels successively for 7,
4 and 3 h in water at 13

C, alternated by air rest stages of

successively 13 and 12 h at 18

C. The imbibed kernels were incu-

bated in the dark at 20

C for up to 24 days. Every 48 h, the kernels

were submerged in water at 20

C during 60 min to prevent

dehydration. During the entire process, the kernels were slowly
rotated and aerated by blowing air at 20

C through the kernel bed.

Two batches of wheat (5 kg each) were processed this way, one
with and one without 10

M GA in the imbibition water.

At the end of the imbibition process and every 24 h thereafter,

samples of approx. 2000 wheat kernel equivalents were collected
from each batch. Half of the sample was frozen immediately in
liquid nitrogen, freeze-dried, weighed and ground using a Cyclotec
1093 sample mill (Tecator, Hog

€an€as, Sweden). The other half was

subjected to surface sterilization as described below.

Extra samples of 500 kernel equivalents were withdrawn at 2, 4,

5, 6, 7, 10, 13, 18, 21 and 24 DPI with GA. For each of these samples,
the roots, coleoptiles and shoots, further referred to as the vege-
tative parts, were separated carefully from the kernel remnants.
The vegetative parts and kernel remnants were separately freeze-
dried, weighed and ground using the Cyclotec mill.

2.3. Surface sterilization

Surface sterilization of the above described samples was per-

formed as described by Dornez et al.

[16]

. To this end, about 1000

kernel equivalents were shaken for 10 min in sodium hypochlorite
[200 mL, 3.0% (w/v) active chlorine] on a horizontal shaker
(Laboshake, VWR International, Leuven, Belgium) at 150 strokes
per 60 s. After decanting the sodium hypochlorite phase, the
samples were rinsed twice with deionised water (800 mL, 10 min),
freeze-dried and ground using the above cited Cyclotec mill.

2.4. Extract preparation

Freeze-dried ground material (2.0 g) was extracted by sus-

pending it in sodium acetate buffer [20.0 mL, 25 mM, pH 5.0, 0.02%
(w/v) sodium azide] and shaking the suspension for 30 min at 7

on the above cited horizontal shaker at 150 strokes per 60 s. After
centrifugation (10 min, 10,000 g, 7

C), the supernatant was

ﬁltered

E. De Backer et al. / Plant Physiology and Biochemistry 48 (2010) 90

e97

through an MN 615

ﬁlter (diameter 90 mm) (MachereyeNagel,

€uren, Germany), yielding the ﬁnal extract.

2.5.

-xylanase activity assay

-xylanase activities in wheat samples were measured colori-

metrically using the Xylazyme-AX method (Megazyme). Extracts
(1.0 mL), prepared as described above, were equilibrated for 10 min
at 40

C before adding a Xylazyme-AX tablet. After appropriate

incubation times at 40

C, the reaction was stopped by addition of

1.0% (w/v) Tris solution (10.0 mL) and vigorous vortex stirring. After
ﬁltration through an MN 615 ﬁlter, the extinction at 590 nm was
measured against a control, prepared by incubating the

ﬁltered

extracts without the substrate tablet at 40

C. In addition, correction

was made for the non-enzymatic colour release from the Xylazyme-
AX tablets. Activities were expressed as milli-

-xylanase units

(mXU) per kernel equivalent, or per vegetative part or kernel
remnant of one kernel equivalent. One

-xylanase unit is the

amount of enzyme needed to increase the absorbance at 590 nm by
1.00 in 60 min at 40

C under the above described assay conditions.

To test the inhibition sensitivity of wheat

-xylanases,

ﬁltered

extracts (1 mL) were preincubated for 30 min at room temperature
with an excess amount of pure TAXI and XIP proteins (13

inhibitor per assay). The

-xylanase activity in the preincubated

samples was then measured with the Xylazyme-AX method as
described above.

2.6. Protein quanti

ﬁcation

Protein concentrations in wheat extracts were estimated

according to Bradford

[5]

with BSA as standard.

2.7. Immunoblotting

2.7.1. SDS-PAGE

Samples were separated by SDS-PAGE in regular slab gels

(80.0 mm

73.0 mm 1.5 mm) consisting of a stacking gel [total

acrylamide 4.0% (w/v)] and a running gel [total acrylamide 12.0%
(w/v)] using the Bio-Rad Mini-Protean 3 Cell electrophoresis unit.
Extracts containing 150

g of protein or low molecular mass

markers were applied on the gels. Separation was performed at
160 V for 90 min at 7

2.7.2. Transfer and immunodetection

Proteins, separated by SDS-PAGE, were transferred onto Protran

nitrocellulose membranes with the Bio-Rad Trans-Blot Semi-
Dry Electrophoretic Transfer Cell at 16 V during 40 min at room
temperature. Immunodetection was performed as described by
Beaugrand et al.

[2]

. Free protein binding sites were blocked with

casein solution [1.0% (w/v)], prepared in 10 mM sodium phosphate
buffered saline (100 mM sodium chloride, 2.0 mM potassium chloride)
at pH 7.4 containing 0.01% (v/v) Tween 20 (referred to as PBS

eT). After

washing the membranes with PBS

eT, they were incubated (60 min,

room temperature) with a 2000-fold dilution of the anti-barley X-I
antiserum in PBS

eT. Thereafter, the membranes were washed again

with PBS

eTand incubated (60 min, room temperature) with a solution

of horseradish peroxidase conjugated goat antibodies against rabbit
IgG as prescribed by the manufacturer of these secondary antibodies.
Subsequently, the membranes were washed again with PBS

eT and

incubated with 3,3

,5,5

-tetramethylbenzidine solution (Sigma-

eAldrich) as substrate for the horseradish peroxidase. After an
appropriate incubation time, the staining was stopped by washing the
membranes in deionised water. Scans of the membranes were made
using an U

max

PowerLook 1120 scanner and MagicScan 4.6 software

(GE Healthcare).

2.8. Analysis of arabinoxylan content and composition

The total AX, WEAX and water unextractable AX (WUAX) levels

and compositions in the above described samples were estimated
by gas chromatography of alditol acetates obtained after acid
hydrolysis of the samples and reduction and acetylation of resulting
monosaccharides as described by Van Craeyveld et al.

[43]

For total AX content the freeze-dried ground samples were

analysed as such, while for WEAX quanti

ﬁcation the extracts of

these samples were used.

AX content was calculated as 0.88 times the sum of the contents

of arabinose and xylose. A correction of the arabinose level for the
presence of water extractable arabinogalactan-peptides based on
an arabinose to galactose ratio of 0.7 was made

[31]

. In doing so, we

assumed that the arabinogalactan-peptide level does not change
throughout germination and initial plant development

[40]

. AX

content is expressed in mg per kernel equivalent, or per vegetative
part or kernel remnant of one kernel equivalent. The data used for
the calculation of total AX, WEAX and WUAX contents also allowed
calculating their A/X ratios.

3. Results

3.1. Wheat germination in the absence and presence of added GA

In the absence of added GA in the imbibition water, the cole-

optile emerged from the seed at 1 DPI. The average length of the
coleoptile increased slowly to 1.5 mm during the

ﬁrst 4 DPI.

Thereafter, it increased at a faster rate to an average of 32 mm at 13
DPI (

Fig. 1

). On average, the

ﬁrst leaf emerged from the coleoptile at

13 DPI (

Fig. 2

). At 24 DPI, the average length of the developing shoot

was 73 mm. Treatment with GA during imbibition had no signi

ﬁ-

cant in

ﬂuence on the time of emergence of the coleoptile and on

the length of the shoot (

Fig. 1

During this growth process, the dry mass per kernel equiva-

lent gradually decreased from 35.1 mg for the unimbibed kernel
to 17.7 mg at 24 DPI (

Fig. 1

). This can be explained by the

increased respiration rate of the kernels, causing metabolic losses
in dry weight. These weight losses made us express

-xylanase

activity and AX levels on kernel equivalent basis instead of on
dry weight basis. Treatment of the wheat kernels with GA during
imbibition had no signi

ﬁcant inﬂuence on the magnitude of the

100

ui i 1 2 3 4 5 6 7 8 9 1011 12131415161718192021222324

)

m (

h t

n e

) t

n e

v i

q e

l e

n r

k /

m (

s s

a
M

Time (DPI)

with added GA

without added GA

with added GA

without added GA

Fig. 1. Dry mass (mg/kernel equivalent), measured per 1000 kernel equivalents, of
germinating wheat kernels or wheat seedlings during 24 days post-imbibition (DPI) in
the presence (

) and absence of added gibberellic acid (GA) (

), and average

length (mm) of coleoptiles or shoots of 30 seedlings at several time points after
imbibition in the presence (

>) and absence of added GA (,) (unimbibed wheat

kernel

¼ ui, imbibed wheat kernel ¼ i).

E. De Backer et al. / Plant Physiology and Biochemistry 48 (2010) 90

e97

metabolic losses (

Fig. 1

). When considering the mass of the

vegetative part and the kernel remnant separately, we found
that, at 24 DPI, the share of the kernel remnant in the total
weight of one kernel equivalent had decreased to an average of
46% (

Table 1

3.2. Endogenous

-xylanases

3.2.1.

-xylanase activity

After imbibition in the absence of added GA,

-xylanase

activity increased very slowly during the

ﬁrst 6 days. It then

increased more steeply until 16 DPI. Finally, it increased almost
exponentially until 24 DPI (

Fig. 3

A). In general,

-xylanase activity

increased from 3 mXU in the unimbibed kernel to 384 mXU per
kernel equivalent after 24 DPI (

Fig. 3

A). The presence of GA in the

imbibition water resulted in an earlier and more drastic increase of

-xylanase activity. The greatest impact of GA addition was

observed between 3 and 12 DPI. More speci

ﬁcally,

-xylanase

activity increased from 9 mXU per kernel equivalent at 3 DPI to 79
mXU at 6 DPI, while in the absence of added GA,

-xylanase

activities were 4 mXU at 3 DPI and 8 mXU at 6 DPI. Following
imbibition with GA,

-xylanase activity remained relatively

constant between 6 and 16 DPI and then increased to 334 mXU per
kernel equivalent after 24 DPI (

Fig. 3

A). After 13 DPI, the

xylanase activity pro

ﬁles of kernels imbibed with and without

added GA more or less converged (

Fig. 3

A). Corder and Henry

[9]

observed for wheat germinated in the absence of added GA an
earlier increase in

-xylanase activity than we observed after

imbibition without added GA. The

-xylanase activity pro

ﬁle

obtained by Caspers et al.

[7]

for barley germinated in the absence

of added GA was comparable to the present activity pro

ﬁle for

wheat germinated without added GA.

Analysis of vegetative parts and kernel remnants showed that

throughout germination and seedling growth the

-xylanase

activity remained concentrated in the kernel remnants (

Table 1

However, whereas Elliott et al.

[17]

did not detect

-xylanase

activity in either roots, shoots or leaves from germinated wheat,
here, low

-xylanase activity was detected in the vegetative part.

Following imbibition with added GA, the share of the

-xylanase

activity in the vegetative parts increased slowly to 5.8% of the total

-xylanase activity at 13 DPI and to 13.5% at 24 DPI (

Table 1

Surface sterilization of the samples with sodium hypochlorite

prior to grinding and the addition of an excess amount of pure TAXI
and XIP to extracts of processed wheat caused no signi

ﬁcant

reduction in

-xylanase activity. These observations con

ﬁrm that

endogenous

-xylanases are not inhibited by these proteinaceous

-xylanase inhibitors and point to very low, if any, contribution of

microbial

-xylanases to the

-xylanase activities measured

under the experimental conditions used in this study.

3.2.2. Occurrence of different wheat

-xylanase forms

Immunoblot analysis using anti-barley X-I PAbs

ﬁrst of all

demonstrated the suitability of the PAbs for detection of wheat X-I
like

-xylanase, as cross-reaction was observed. This was antici-

pated in view of the very high similarity in amino acid sequence
between both enzymes

[38,37]

(88% identity, 93% similarity).

Moreover, several

-xylanase forms appeared which, in analogy

Fig. 2. Wheat germination (until day 13) and seedling development (day 13 to day 24) following imbibition without added gibberellic acid. Photographs were taken after 2, 4, 5, 6, 7,
10, 13, 18, 21 and 24 days post-imbibition (DPI).

Table 1
The share (%) of mass and

-xylanase activity in the vegetative part and the kernel

remnant in the dry mass and activity of the intact sample during germination and
seedling growth following imbibition with added gibberellic acid.

Dry mass (%)

-xylanase activity (%)

2 days post-imbibition

kernel remnant

98.3

97.8

vegetative part

1.7

2.2

4 days post-imbibition

kernel remnant

95.3

96.1

vegetative part

4.7

3.9

5 days post-imbibition

kernel remnant

93.4

94.8

vegetative part

6.6

5.2

6 days post-imbibition

kernel remnant

89.8

96.8

vegetative part

10.2

3.2

7 days post-imbibition

kernel remnant

87.8

96.9

vegetative part

12.2

3.1

10 days post-imbibition

kernel remnant

80.7

95.9

vegetative part

19.3

4.1

13 days post-imbibition

kernel remnant

71.9

94.2

vegetative part

28.1

5.8

18 days post-imbibition

kernel remnant

57.4

88.3

vegetative part

42.6

11.7

21 days post-imbibition

kernel remnant

48.8

89.8

vegetative part

51.2

10.2

24 days post-imbibition

kernel remnant

46.4

86.5

vegetative part

53.6

13.5

E. De Backer et al. / Plant Physiology and Biochemistry 48 (2010) 90

e97

to barley

[7]

, probably originate from the same parent X-I like

precursor, as they were all recognized by the PAbs (

Fig. 3

B and C).

The blots furthermore showed an increase in the level of

endogenous

-xylanase during germination and seedling growth

(

Fig. 3

B and C). With no GA added to the imbibition water, a signal

corresponding to a 67 kDa wheat

-xylanase appeared very

weakly at approx. 6 DPI and further intensi

ﬁed until 24 DPI. Pro-

cessed

-xylanase forms of 50 kDa, 43 kDa and 34 kDa appeared

clearly at approx. 18 DPI (

Fig. 3

B) and became more prominent at

longer germination times. Addition of GA to the imbibition water
not only increased

-xylanase activity between 3 and 12 DPI

(

Fig. 3

A), but also resulted in a strongly increased abundance of the

67 kDa

-xylanase form before 13 DPI.

At the same sample withdrawal times, kernel remnants and

non-dissected material showed on immunoblot

-xylanase bands

of comparable intensities. In the vegetative part, the unprocessed

-xylanase and the different forms derived from it could not be

detected (results not shown), although low but increasing

xylanase activities were measured as discussed above.

3.3. Arabinoxylan content and extractability

The level and extractability of AX during germination and

subsequent seedling growth were studied in relation to the
increasing endogenous

-xylanase activity. The total AX and

WUAX levels in the imbibed wheat kernels were lower than in
the unimbibed ones (

Fig. 4

). This was possibly caused by partial

(kDa)

67.0

43.0

30.0

20.1

LMW 4 DPI 6 DPI 10 DPI 13 DPI 18 DPI 21 DPI 24 DPI

(kDa)

67.0

43.0

30.0

20.1

100

150

200

250

300

350

400

450

ui i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

yti

/kernel

Time (DPI)

with added GA

without added GA

Fig. 3.

-xylanase activity (mXU/kernel equivalent) in unimbibed wheat kernels (ui),

imbibed wheat kernels (i), and germinating wheat kernels or wheat seedlings during
a 24 day period after imbibition in the presence (

) and absence of added gibberellic

acid (GA) (

) (A). Immunoblots of wheat extracts during germination and seedling

growth after imbibition without (B) and with added GA (C). For each sample 150

g of

protein was loaded on SDS-PAGE gel. Lane 1: Low molecular weight (LMW) markers; lane
2

e8: 4, 6, 10, 13, 18, 21 and 24 days post-imbibition (DPI), respectively. The molecular

mass (MM) of the LMW markers are indicated on the left side.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

ui i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A l

Time (DPI)

with added GA

without added GA

0.0

0.1

0.2

0.3

0.4

0.5

ui i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

WEAX content

Time (DPI)

with added GA

without added GA

0.0

0.5

1.0

1.5

2.0

2.5

3.0

ui i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

U
W

equivalent)l

Time (DPI)

with added GA

without added GA

Fig. 4. Total arabinoxylan (total AX) content (A), water extractable arabinoxylan
(WEAX) content (B) and water unextractable arabinoxylan (WUAX) content (C) (mg/
kernel equivalent) in unimbibed wheat kernels (ui), imbibed wheat kernels (i), and
germinating wheat kernels or wheat seedlings during 24 days post-imbibition (DPI) in
the presence (

) and absence of added gibberellic acid (GA) (

E. De Backer et al. / Plant Physiology and Biochemistry 48 (2010) 90

e97

removal of outer layers from the wheat grains by friction between
the kernels due to washing and rotation during imbibition. From 1
DPI until 7 DPI, the total AX and WUAX levels remained almost
constant at approx. 1.9 and 1.6 mg per kernel equivalent, respec-
tively, and then increased to 2.8 and 2.6 mg per kernel equivalent at
24 DPI, respectively (

Fig. 4

A and C). When GA was added, a trend

towards lower, but still increasing total AX and WUAX levels with
longer germination times was observed. After imbibition without
GA, the WEAX level increased from 0.15 mg to 0.35 mg per kernel
equivalent before 9 DPI and then decreased from 13 DPI, at emer-
gence of the

ﬁrst leaf, until 24 DPI. The initial increase in WEAX

level prior to a decrease has also been described for malting barley

[40]

. Imbibition with added GA accelerated the increase in WEAX

content, but the absolute maximum level was similar to that
without GA added during imbibition. In particular, the WEAX level
increased to 0.38 mg per kernel equivalent at 5 DPI and decreased
from 8 DPI until 24 DPI (

Fig. 4

B). The A/X ratios of total AX, WEAX

and WUAX did not change signi

ﬁcantly during germination and

seedling growth.

Analysis of AX in different plant parts revealed that the total AX

level decreased from 2.1 mg to 1.3 mg per kernel remnant at 13 DPI
with added GA and remained almost constant thereafter (

Fig. 5

A).

A similar trend has already been observed by others during barley
malting

[26]

. It is remarkable that no, or only very little, AX

solubilisation was observed following 5 DPI, given that from 16 DPI
onwards a large increase in

-xylanase activity, mostly located in

the kernel remnants (

Table 1

), was detected (

Fig. 3

). The WEAX

level was maximal at 0.40 mg per kernel remnant after 5 DPI with
added GA, while the WUAX level decreased during the

ﬁrst 5 DPI

and then remained more or less constant at approx. 1.2 mg per
kernel remnant (

Fig. 5

A). The changes in WEAX level in the kernel

remnants very much resembled those measured in the intact
samples.

In the vegetative parts, the total AX and WUAX levels gradually

increased to 1.37 mg and 1.32 mg per kernel equivalent, respec-
tively, at 24 DPI with added GA, while the WEAX level remained
very low (

Fig. 5

B). The increase in the total AX and WUAX levels is

in line with the increase in dry mass of the vegetative parts
(

Table 1

). The A/X ratios of total AX and WUAX decreased (

Fig. 5

C).

4. Discussion

Germination starts with the uptake of water by the wheat

kernels and then leads to mobilisation of the endosperm reserves
and the development of the embryo into a plant. During germi-
nation and seedling growth, endogenous

-xylanase activity

drastically increased, while, at the same time, a strong increase in
the occurrence of 67 kDa wheat

-xylanase and subsequent

processing of this enzyme to forms of approx. 50 kDa, 43 kDa and
34 kDa was detected (

Fig. 3

). Based on these results and the data on

AX solubilisation, we propose that roughly three different stages
(early, intermediate and late) can be discriminated during wheat
germination and seedling growth. These stages strongly differ in
the occurrence of

-xylanases (type, processing) and

-xyla-

nase activity.

In the early stage (up to 3

e4 DPI), low but signiﬁcant

xylanase activities are detected whether or not GA is added to the
imbibition water (

Fig. 3

A). Furthermore, the WEAX level in the

wheat kernel remnants strongly increases while the WUAX level
decreases (

Fig. 5

A), indicating solubilisation of WUAX in the aleu-

rone and endosperm cell walls by means of

-xylanases

[29]

However, this is not accompanied by the occurrence of a

xylanase band on immunoblot (

Fig. 3

B and C). Hence, wheat X-I and

X-II like

-xylanases, i.e. homologues of the barley X-I and X-II

enzymes, are probably not involved in the loosening of endosperm

and aleurone cell walls in the early stage of germination since they
would probably be detected with the anti-barley X-I PAbs. This
indicates that new, yet unidenti

ﬁed,

-xylanases are probably

involved in endosperm and aleurone cell wall loosening. Compa-
rable results were obtained by Caspers et al.

[7]

for barley. The

presence of different enzymes in the early stage of germination was
recently also described for an endo-

-mannanase system during

rice germination

[34]

. The early stage endo-

-mannanases have

higher isoelectric points and carbohydrate binding domains,
enabling them to attach to cell walls. Poor extractability of the
hypothetical early stage

-xylanases due to strong interaction

with cell walls under the conditions used could be an explanation
for the low activities measured, while the WEAX level strongly
increases. The faster AX solubilisation, when GA was added to the

0.0

0.5

1.0

1.5

2.0

2.5

ui i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Time (DPI)

Total AX

WEAX

WUAX

0.0

0.5

1.0

1.5

2.0

2.5

ui i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

r l

Time (DPI)

Total AX

WEAX

WUAX

0.0

0.2

0.4

0.6

0.8

1.0

1.2

ui u 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Time (DPI)

Total AX

WEAX

WUAX

Fig. 5. Total arabinoxylan (total AX) (

), water extractable arabinoxylan (WEAX)

(

) and water unextractable arabinoxylan (WUAX) (

) levels (mg/kernel

equivalent) in wheat kernel remnants (A) and vegetative parts (B) and the arabinose to
xylose (A/X) ratios (C) of total AX (

), WEAX (

) and WUAX (

) in wheat

vegetative parts during 24 days post-imbibition (DPI) in the presence of added gib-
berellic acid (GA).

E. De Backer et al. / Plant Physiology and Biochemistry 48 (2010) 90

e97

imbibition water, was only visible in this stage and resulted in
higher WEAX levels.

In the intermediate stage (up to 13 DPI), increased

-xylanase

activity goes hand-in-hand with an increased level of the unpro-
cessed 67 kDa

-xylanase, which is more pronounced in the

presence of GA during imbibition (

Fig. 3

C). In addition, WUAX in the

kernel remnants are further solubilised as far as possible and can be
mainly ascribed to degradation of AX in the aleurone and endo-
sperm cell walls

[29]

while the WEAX level reaches a maximum

(

Fig. 5

A). This strongly suggests that the 67 kDa

-xylanase form

is active in vivo, in line with results reported by Van Campenhout
et al.

[41]

. These authors convincingly demonstrated that the

unprocessed recombinant barley X-I enzyme is functional.
According to Caspers et al.

[7]

aleurone PCD is a prerequisite for

releasing this enzyme. Taken together with the knowledge that GA
addition stimulates aleurone PCD

[15]

or PCD of other cells

[12]

, this

allows us to propose that GA addition stimulates both wheat X-I
like

-xylanase synthesis and the release of the unprocessed

enzyme. However, the question can be raised as to why little if any
processing occurs in the intermediate stage. It can be speculated
that the proteases involved in the processing are not yet present in
suf

ﬁcient amounts or stored in protein storage vacuoles, suggesting

that their action might not be directly regulated by GA but rather as
a function of development. Such kind of regulation would assure
a keen

ﬁne-tuning and a gradual increase in

-xylanase activity.

Indeed, when considering the evolution of

-xylanase activity

and the brightness of the bands on immunoblot, one can expect
a lower speci

ﬁc activity for unprocessed

-xylanase than for the

processed forms. This should be further studied by activity and
substrate speci

ﬁcity measurements on the separate 67, 50, 43 and

34 kDa enzyme forms.

In the late stage, further wheat

-xylanase processing can be

related to strongly increased

-xylanase activities (

Fig. 3

), in line

with earlier observations for barley

[7]

. The processed enzyme

forms probably play an essential role in the extensive hydrolysis of
aleurone and endosperm cell walls and the recycling of carbon
skeletons. It is likely that the unprocessed

-xylanase form binds

to easily accessible AXs by means of its carbohydrate binding
module, whereas the processed

-xylanase forms, having lost this

module and decreased in molecular mass, become more mobile
and are able to hydrolyse AXs embedded in more complex cell wall
environments

[41]

During the intermediate and late stages, not all WUAX in the

kernel remnants are solubilised although xylanase activity
increases. However, the A/X ratio (approx. 0.55) of the remaining
AXs in the remnants should have allowed further

-xylanase

action. This suggests that the occurrence of other substituents, e.g.
uronic acid and acetyl moieties in glucuronoarabinoxylans, and
cross linking with other cell wall constituents, e.g. lignin, in
complex cell wall environments may hamper AX degradation. This
is probably the case in the outer grain tissues that are left as an
empty pocket at the end of germination (

Fig. 2

). However, the steep

increase in

-xylanase activity and

-xylanase processing

(

Fig. 3

) is concomitant with the decrease in WEAX level in the

kernel remnants (

Fig. 5

A). The degradation products of WEAX serve

the seedling's needs for e.g. energy and building blocks

[10]

Furthermore, the total AX level and the WUAX level in the

vegetative parts increase due to deposition of AX in the newly
synthesized cell walls of the developing seedling

[23]

. The

decreasing A/X ratios (

Fig. 5

C) should facilitate AX incorporation

into the cell wall

[29,6,22,23]

Although the highest

-xylanase activities were detected in

the kernel remnants, low

-xylanase activities were also

measured in the vegetative parts of the developing seedling. This
may be explained by the presence of one or more

-xylanases in

the vegetative parts that assist in the remodelling of cell wall AXs
during cell expansion. Such presence was earlier suggested for
other plant

-xylanases such as the barley X-II

-xylanase

[38,42]

. However, on immunoblot no

-xylanases were recog-

nized by the PAbs against barley X-I

-xylanase suggesting that

-xylanases present in the vegetative part are not directly related

to those present in the wheat kernel remnant.

In conclusion, the results described here demonstrate the

occurrence of three stages during wheat germination and seedling
growth which can be discriminated from one another based on the
occurrence of different endogenous

-xylanases and their activity

in situ on wheat AX. In addition to the X-I like

-xylanase, which

plays a crucial role in the intermediate and late stages, the results
suggest that additional

-xylanases, presumably with low

homology to the X-I enzyme, are essential during germination and
seedling development.

Acknowledgements

Kurt Gebruers is a postdoctoral fellow of the Fonds voor

Wetenschappelijk

Onderzoek-Vlaanderen

(FWO-Vlaanderen,

Brussels, Belgium). Wim Van den Ende is supported by grants from
FWO-Vlaanderen. The study was in part carried out in the frame-
work of research project GOA/03/10

ﬁnanced by the Research Fund

K.U. Leuven and is part of the K.U.Leuven Methusalem programme
“Food for the Future”. The authors thank Prof. Maarten Chrispeels
(University of California San Diego (UCSD)) for critical discussions.

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Document Outline

Post-translational processing of beta-d-xylanases and changes in extractability of arabinoxylans during wheat germination

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