Journal of Cereal Science 37 (2003) 187±194
doi:10.1006/jcrs.2002.0493
A Wheat Xylanase Inhibitor Protein (XIP-I)
Accumulates in the Grain and has Homologues in
Other Cereals
G. O. Elliott, W. R. McLauchlan, G. Williamson and P. A. Kroon*
Diet, Health and Consumer Sciences Division, Institute of Food Research,
Colney Lane, Norwich NR4 7UA, UK
Received 25 January 2002
ABSTRACT
We have measured xylanase inhibitor activity against an Aspergillus niger xylanase in different parts of the
wheat plant at different stages of development and used immunodetection to determine the spatial and
temporal distribution of xylanase inhibitor protein I (XIP-I) in Triticum aestivum var. Soisson. Although
xylanase inhibitor activity was detected in all parts of the wheat plant throughout development, XIP-I
was located predominantly in the grain tissue, where it appears at a late stage in grain development and
persists after germination, indicating that the different xylanase inhibitor proteins are under different
regulatory controls. (1,4)-b-Xylanase activity was detected in wheat grains during development and postgermination. Pure XIP-I and a crude sample containing TAXI inhibitors but not XIP-I did not have the
ability to inhibit this endogenous (1,4)-b-xylanase activity. Protein extracts from the seeds of various
monocots were also tested for the presence of XIP-I, where it was found to be present in the grains of
several wheat varieties, rye and barley, but was not detected in rice, sorghum or maize.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Triticum aestivum, Hordeum vulgare, Oryza sativa, Secale cereale, immunodetection, endo-1,4-b-D-xylanase.
INTRODUCTION
The ®rst reports indicating that cereal seeds contain
inhibitors of endo-1,4-b-D-xylanase ((1,4)-b-xylanase) activity were published in the late 1990s1±4.
They described the inhibition of (1,4)-b-xylanase
activities in the presence of wheat ¯our protein
extracts, and demonstrated that the inhibitory activity was heat-sensitive. Subsequently, we described
the isolation of a unique xylanase inhibitor protein
(XIP-I) from wheat ¯our5. This 29 kDa protein has a
ABBREVIATIONS
USED:
XIP-I ˆ Xylanase
Inhibitor
Protein-I; TAXI ˆ Triticum aestivum L. xylanase inhibitor;
kDa ˆ Kilo Daltons; DTT ˆ dithiolthreitol; SDS ˆ
sodium dodecyl sulphate; DNS ˆ dinitrosalicylic acid.
This work was funded by the Biotechnology and
Biological Sciences Research Council (UK) and Brewing
Research International (CASE studentship to GOE).
* Corresponding Author: E-mail: [email protected]
0733±5210/03/020187 ‡ 08 $35.00/0
pI of 87±89, and inhibited reversibly, in a competitive manner, family 11 (1,4)-b-xylanases from
Aspergillus niger and Trichoderma viride. A second protein
termed Triticum aestivum xylanase inhibitor (TAXI)
that exhibited inhibitory activity towards (1,4)-bxylanases was isolated from wheat ¯our6. It was
subsequently shown that two forms of TAXI are
present in wheat (denoted TAXI-I and TAXI-II),
and that these two forms can be distinguished by
different inhibitory activities towards various (1,4)-bxylanases and by their different pI values (88 and
93 respectively)7. Recently a homologue of
TAXI was identi®ed in barely, which was termed
Hordeum vulgare L, xylanase inhibitor (HVXI)8.
It has been shown that TAXI has a deleterious
effect on loaf volume in baking, and it was suggested
that this was due to inhibition of endogenous (1,4)-bxylanase activity6. The use of microbial (1,4)-bxylanases in industrial processes is widespread and
# 2003 Elsevier Science Ltd. All rights reserved.
188
G. O. Elliott et al.
the potential for inhibitors of this activity to have
detrimental effects is therefore great.
To date, xylanase inhibitor proteins have been
identi®ed only in ¯our, and nothing is known concerning their biosynthesis in the developing grain.
Further, it is not known if proteinaceous xylanase
inhibitors are active towards endogenous (1,4)-bxylanases, and the existence of XIP-I homologues in
cereals outside of wheat has not been assessed. We
have addressed these issues by measuring xylanase
inhibitor activity against an A. niger xylanase in
various tissues throughout the development of
wheat plants, and have screened for the presence
of XIP-I using speci®c polyclonal antibodies. We
have also determined if puri®ed XIP-I or a crude
sample containing TAXI proteins were able to
inhibit endogenous (1,4)-b-xylanase activity, and
tested other cereal grains for both inhibitor activity
and the presence of XIP-I protein.
inhibitor cocktail (CompleteTM ; Boehringer
Mannheim GmbH, Germany; one tablet per 50 ml
extraction buffer). Suspensions were homogenized
using a Polytron1 PT 1300 D homogeniser
(Kinematica AG, Luzern, Switzerland). The slurry
was centrifuged at 10 000 g for 3 min at 4 C and
the supernatant was collected. Crude extracts were
desalted using a disposable column pre-packed with
Sephadex G-25 (NAP-5; Amersham Pharmacia
Biotech, UK) prior to use.
MATERIALS AND METHODS
SDS-PAGE
Plant material
Desalted and reduced (50 mM DTT) protein extracts
were electrophoresed in the presence of SDS using
10% Bis-Tris NuPAGE gels and the MOPS buffer
system (Novex, Frankfurt, Germany) according to
the manufacturers instructions. Molecular weights
were calculated from a plot of migration distance
versus log10 of the molecular weight for a series of
biotinylated protein markers (broad range; New
England BioLabs, Hitchin, UK).
Grain samples were kind gifts from the John Innes
Centre, Norwich, UK; T. aestivum var. Huntsman,
T. aestivum var. Soisson, T. aestivum var. Chinese
spring, T. monococcum, T. durum var. Cadpur, Secale
cereale var. King II, and Oryza sativa TN1. H. vulgare
var. Halcyon was supplied by Brewing Research
International, Nut®eld, Surrey. Wheat ¯our (ex.
T. aestivum var. Soisson) was a kind gift from
Unilever, Vlaardingen, the Netherlands.
Wheat (T. aestivum var. Soisson) was grown in a
glasshouse (12 C, 15 h light/day). Seeds were
germinated on paper soaked in distilled H2O in
the dark at 25 C for 48 h before being transferred to
soil pots in a glasshouse. Except for developing grain,
day zero was taken as imbibition. For developing
grain, day zero was when emergence of ear was
complete (stage 105 on the Feekes scale). Samples
were taken at the time-points indicated in the results
section, snap frozen in liquid nitrogen and stored
at ÿ80 C.
Preparation of protein extracts
Samples (generally 1 g fresh weight) were ground to a
®ne powder under liquid nitrogen using a pestle and
mortar and subsequently mixed with 2 volumes of
McIlvaines buffer (01 M citrate/02 M sodium
phosphate, pH 55) which also contained a protease
Assay of total protein
Total protein was assayed using the Coomassie Plus
protein assay reagent (Pierce & Warriner, Chester,
UK), which is based on the method of Bradford9,
using bovine serum albumin (Sigma-Aldrich, Poole,
UK) for the standard curve.
Measurement of (1,4)-b-xylanase activity
(1,4)-b-xylanase activity was determined using a
dinitrosalicylic acid (DNS)-based colourimetric
assay for reducing sugars10. The substrate [100 ml
of 2% birchwood (1,4)-b-xylan (Roche, Lewes, UK)
in McIllvaines buffer] and an appropriate volume of
buffer were equilibrated at 30 C. Crude protein
extract in McIlvaines buffer was added to give a ®nal
volume of 200 ml, reactions were incubated at 30 C
for 1 h, and were terminated by the addition of
300 ml of DNS solution. Samples were boiled (5 min)
and allowed to cool before centrifugation at
13 000 g (2 min). The absorbance was determined
at 550 nm relative to a xylose standard curve (0±
180 mg/mL). Assays were performed in triplicate
along with appropriate blanks. One unit of (1,4)-bxylanase activity was de®ned as the amount of
protein which released 1 mmol of xylose minÿ1.
Accumulation of a wheat xylanase inhibitor protein
The assay was linear over the range 0±08 absorbance units (AU), with a detection limit of 002 AU.
Assay for xylanase inhibitor activity
Inhibition of (1,4)-b-xylanase activity was determined by comparing the reduction in (1,4)-bxylanase activity in the presence of wheat
protein extracts compared to appropriate controls,
which were incubated in the absence of wheat
extracts. A known concentration of an endo-1,4-bD-xylanase from Aspergillus5 (see McLauchlan et al.,
1999) was incubated with birchwood (1,4)-b-xylan in
the presence or absence of wheat protein extracts
using the DNS assay described in the preceding
section. Crude protein extracts (®nal diluted volume
94 ml) were pre-incubated (5 min, 30 C) with xylanase (6 ml 277 ng). Reactions (in triplicate) were
initiated by the addition of pre-warmed (30 C) 2%
birchwood (1,4)-b-xylan (100 ml) and terminated
with DNS reagent (300 ml) after an appropriate
incubation period (usually 10 min). One unit of
inhibitor activity was de®ned as the amount of
inhibitor that caused a reduction in absorbance at
550 nm of 01 AU in respect to (1,4)-b-xylanase
controls under the conditions described. As it was
demonstrated that (1,4)-b-xylanase activity was present in some of the crude protein extracts (developing and germinating grain), it was necessary to take
into account endogenous (1,4)-b-xylanase activity
when estimating the amount of xylanase inhibitor
activity in these samples. Accordingly, for developing grain and germinating grain samples the change
in absorbance at 550 nm was corrected by subtracting the change in absorbance due to endogenous
(1,4)-b-xylanase activity before inhibition activity
was calculated.
Isolation of wheat xylanase inhibitor proteins
and production of polyclonal antibodies
XIP-I was puri®ed from wheat (var. Soisson) ¯our
according to the method of McLauchlan et al.5, and
the purity con®rmed by SDS-PAGE (see above).
During puri®cation, two inhibitor activities were
observed; the second activity was separated from
XIP-I since it did not bind to a DEAE-Sepharose
column under the conditions used. This crude
xylanase inhibitor-containing fraction was used in
subsequent experiments as the source of XIP-I-free
xylanases inhibitor activity. Anti-XIP-I polyclonal
antibodies were produced by injecting 4-month-old
189
male New Zealand White rabbits with 100 mg of pure
XIP-I mixed 1:1 with Freund's complete adjuvant in
a ®nal volume of 100 ml. Similar injections were
given at 5 weeks and 8 weeks again using 100 mg
XIP-I per injection, mixed 1:1 with Freund's incomplete adjuvant. Rabbits were bled 10 days and 14
days after the ®nal injection. Blood was collected in
heparinised tubes and immediately centrifuged
(5000 g, 10 min) to recover plasma that was stored
in aliquots (100 ml) at ÿ20 C.
Immunodetection
Following electrophoresis, gels were blotted onto
02 mm nitrocellulose membranes (BioRad) as
described elsewhere11. Subsequently, membranes
were blocked for 1 h at room temperature with
TBS/Tween (50 mM Tris, pH 74/200 mM NaCl/
01% Tween 20) containing 5% dried milk powder.
Membranes were then incubated with the primary
antibody (anti-XIP-I, 1:5000 dilution) in TBS/
Tween containing 1% milk powder for 1 h at room
temperature before being washed three times in TBS/
Tween, once vigorously and twice for 5 min on a
rotary shaker. Membranes were then hybridised with
the secondary antibody [goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich),
1:2000 dilution] in TBS/Tween containing 1% milk
powder for 1 h at room temperature. After a further
three washes, blots were developed using chemiluminescent detection reagents (ECL Plus Detection
Kit; Amersham Pharmacia Biotech) according to the
manufacturer's instructions. The detection limit
using pure XIP-I protein was 20 ng per lane.
RESULTS
Endogenous (1,4)-b-xylanase activity
(1,4)-b-xylanase activity was measured in developing
and post-germinating grain samples, and in samples
obtained from root, shoot or leaf. Activity was
detected in whole grains both during grain development and post-germination, but was not detected
in any other part of the developing wheat plant. In
post-germinating grain, mean (1,4)-b-xylanase activity increased from 0086 U (mg protein)ÿ1 in the dry
grain to 065 U (mg protein)ÿ1 seven days after imbibition and then fell slightly to 058 U (mg protein)ÿ1
14 days after imbibition [Fig. 1(a)]. In developing
grain, low amounts (1,4)-b-xylanase activity could be
detected for the ®rst seven days post-emergence,
after which the activity increased up to day 21 before
falling again up to the fourth week [Fig. 1(b)].
X y lan a s e a c tiv i ty [U (mg p r o t e i n–1 )] X y lan a s e a c tiv i ty [U (mg p r o t e i n–1 )]
190
G. O. Elliott et al.
SDS-PAGE analysis of the pooled fractions generated more than one band upon staining with
Coomassie blue, this fraction was free of XIP-I
(lack of reaction with anti-XIP-I polyclonal antibodies). When samples of the XIP-I-free xylanase
inhibitor preparation were incubated in the presence
of (1,4)-b-xylan (substrate) and protein extracts
obtained from germinating T. aestivum grains, we
did not observe a reduction in xylanase activity
compared to control incubations.
(a)
0 .6
0 .4
0 .2
0 .0
(b)
0 .4
Inhibition of Aspergillus (1,4)-b-xylanase by
crude protein extracts from wheat
0 .3
0 .2
0 .1
0 .0
0
5
10
15
20
25
D a ys p o st-g e rm in a tio n (a) o r -a n th e s is (b)
Figure 1 Levels of xylanase activity in germinating (a) and
developing (b) grains of T. aestivum var. Soisson. Xylanase
activity was determined by incubating appropriate volumes of
crude protein extracts with a birchwood (1,4)-b-xylan substrate
at pH 55 and 30 C and measuring the increase in total
reducing groups using a colourimetric assay. Error bars indicate
the standard deviation (n ˆ 3). One unit of xylanase activity is
the amount of enzyme that gives an increase in reducing groups
equivalent to one mmol of xylose per minute.
Inhibition of endogenous xylanase activity by
T. aestivum xylanase inhibitor proteins
Although (1,4)-b-xylanase activity is present in
grains post-germination and during development,
it is not known if the enzyme(s) are susceptible to
inhibition by T. aestivum XIP-I. When protein
extracts obtained from germinating grain [day 7;
see Fig. 1(a)] or developing grain [day 21; see
Fig. 1(b)] were incubated with increasing amounts
of pure wheat XIP-I (0±16 mg), no inhibition of
the (1,4)-b-xylanase activity in either sample was
observed, even at the highest inhibitor concentration
(equivalent to 021 mM XIP-I in the assay). During
puri®cation of XIP-I from wheat ¯our, we observed
a second peak of xylanase inhibitor activity, which
was separated from those fractions corresponding to
XIP-I during anion-exchange chromatography on
DEAE-sepharose (results not presented). Although
We were able to detect the presence of xylanase
inhibitor activity in protein extracts obtained from
roots, shoots, leaves, germinating grain and developing grain for Soisson wheat. When protein extracts
were boiled prior to incubation with (1,4)-b-xylan
and A. niger (1,4)-b-xylanase, the ability to inhibit the
exogenous (1,4)-b-xylanase was lost, con®rming the
proteinaceous nature of the inhibitor(s). For each
sample, the units of inhibitor activity per gram of
tissue (wet weight) were calculated [Figs 2(a)±(e)].
The range of inhibitor units varied from tissue to
tissue, with the highest values being obtained from
the later stages of grain development [Fig. 2(e)], and
the lowest values in the older root samples [Fig. 2(b)].
Immunodetection of XIP-I in crude protein
extracts from Soisson wheat
Polyclonal antibodies raised against puri®ed XIP-I
were used to determine the presence of XIP-I in
crude protein extracts obtained from developing
Soisson wheat plants. To determine the speci®city of
the anti-XIP-I antibodies, 5 mg of crude wheat ¯our
extract (ex. Soisson) were subjected to immunoblotting using the anti-XIP-I serum as the source of
primary antibody and pure XIP-I as a positive
control. In the test lane, we observed a single band
of apparent molecular mass 285 kDa which corresponded with the single band in the lane containing
pure XIP-I, indicating the antiserum was speci®c for
the XIP-I protein (data not presented).
When 20 mg of total protein from crude extracts of
germinating and post-germinating grain were analysed using the anti-XIP-I antibody, we were able to
detect XIP-I in the 0, 2, 4 and 7-day samples, but not
in the 14 day sample (Fig. 3). XIP-I was also detected
in crude extracts obtained from grains at the grain
75
50
25
0
15
5
10
Days post-germination
(e)
4000
100
50
0
2000
0
0
10
20
30
40
Days of anthesis
0(c)
20
40
60
80
(d)
20
40
60
80
20
40
60
80
100
50
0
-1
Inhibitor activity [U (g tissue)-1]
0
191
(b)
-1
(a)
-1
100
Inhibitor activity [U (g tissue) ] Inhibitor activity [U (g tissue) ] Inhibitor activity [U (g tissue) ]
-1
Inhibitor activity [U (g tissue) ]
Accumulation of a wheat xylanase inhibitor protein
0
100
50
0
0
Days post-germination
Figure 2 Xylanase inhibitor activity in protein extracts from developing wheat plants (T. aestivum var. Soisson). Appropriate volumes
of crude protein extracts prepared from the plant tissues shown were incubated in the presence of birchwood (1,4)-b-xylan and a
xylanase from A. niger (as described in Materials and Methods). Inhibitor activities are expressed as U (mg tissue)ÿ1. One unit (U) of
inhibitor activity was de®ned as the amount of inhibitor that reduced the absorbance change at 550 nm by 01 absorbance units
relative to xylanase-only controls. a, germinating grain; b, roots; c, shoots; d, leaves; e, developing grain.
soft dough (35 days; 112 on the Feekes scale) and
after ripening at the grain hard (42 days; 114) stages
of development, but not in extracts obtained from
grain during earlier stages of development (Fig. 3).
XIP-I could not be detected in any other part of the
plant when undiluted extracts were analysed.
Inhibitor activity and immunodetection of
XIP-I in other wheat varieties and cereals
We also measured xylanase inhibitor activity against
an A. niger xylanase in other varieties of wheat and a
number of other cereals. Protein extracts from the
following types of wheat grain were used: T. aestivum
var. Huntsman, T. aestivum var. Chinese spring,
T. monococcum and T. durum. The data presented in
Figure 4 indicate that xylanase inhibitor activity was
present in samples from all of the wheat varieties
tested. The overall levels of inhibitor activity are
comparable to those observed for T. aestivum var.
Soisson, with the highest concentrations present in
extracts obtained from the Huntsman variety and
the lowest in those extracts from T. monococcum and
T. durum. We also found that inhibitor activity was
present in relatively high concentrations in rye
(Secale cereale var. King II) (as previously reported5)
and barley (H. vulgare var. Halycon), but was not
detectable in rice (O. sativa TN1).
192
G. O. Elliott et al.
(a)
(b)
Figure 3 Detection of XIP-I in protein extracts obtained
during germination and development of grains from T. aestivum
var. Soisson. Samples of crude protein extracts (20 mg each)
obtained from developing or germinating grain were electrophoresed under reducing conditions in the presence of SDS,
blotted to nitrocellulose, and probed for the presence of XIP-I
using a polyclonal anti-XIP-I antibody. Lane 1, molecular
weight markers; lanes 2±9, developing grain samples corresponding to 2, 4, 7, 14, 21, 28, 35 and 42 days post-anthesis;
lane 10, pre-germinated grain; 11±14, germinating grain
samples corresponding to 2, 4, 7, and 14 days, respectively;
lane 15, puri®ed XIP-I (50 ng).
Inhibitor activity [U (g tissue)-1]
100
75
50
25
Barley
Rice
Rye
Durum
Monococcum
Chinese Spring
Huntsman
Soisson
0
Wheat varieties
Figure 4 Xylanase inhibitor activity in the grains of various
species of Triticum and in other monocots. Appropriate volumes
of crude protein extracts prepared from the grains of various
Triticum and a selection of other monocots were incubated in the
presence of birchwood (1,4)-b-xylan and a xylanase from A. niger.
One unit of inhibitor activity was de®ned as the amount of
inhibitor that reduced the absorbance change at 550 nm by
01 absorbance units relative to xylanase-only controls.
Figure 5 Probing for the presence of XIP-I in protein extracts
obtained from the grains of various species of Triticum and in
other monocots. The general procedure used is described
in Figure 3. Lanes as follows: (a) 1, molecular weight markers
(see Fig. 2); 2, T. aestivum var. Soisson (28 mg total protein); 3,
T. aestivum var. Huntsman (48 mg); 4, T. aestivum var. Chinese
Spring (48 mg); 5, T. monococcum (48 mg); 6, T. durum (48 mg); 7,
S. cereal (48 mg); 8, O. sativa (48 mg); 9, H. vulgare (48 mg); 10,
puri®ed T. aestivum XIP-I (50 ng), (b) 1, H. vulgare (96 mg); 2,
H. vulgare (48 mg); 3, O. sativa (96 mg); 4, O. sativa (48 mg); 5,
puri®ed T. aestivum XIP-I (50 ng).
Western blot analysis indicated that XIP-I was
present in all the varieties of wheat tested [Fig. 5(a)],
with the highest cross-reactivity observed for samples
from T. aestivum var. Huntsman and Chinese spring,
with lower cross-reactivity observed for samples
from T. monococcum and T. durum. For all wheat
varieties tested, the cross reaction resulted speci®cally in bands with molecular weights at or slightly
lower than 30 kDa, similar to the results obtained
with T. aestivum var. Soisson. Analysis of proteins
extracted from the Huntsman and Chinese spring
varieties of T. aestivum repeatedly revealed three
bands corresponding to molecular weights of 285,
29 and 30 kDa (similar results were obtained with
T. aestivum var. Soisson). However, analysis of proteins extracted from grains of T. monococcum or
T. durum revealed only two distinct bands (285 and
29 kDa). For non-wheat monocots, a cross-reaction
with anti-XIP-I antibodies was observed for rye,
under the same conditions used for wheat (i.e. 50 mg
of total protein per lane), but not in barley or rice
[Fig. 5(a)]. However, when the amount of total
protein loaded onto the gel was doubled to 100 mg
cross reaction with the polyclonal anti-XIP-I
antibody was observed with barley, but not with
rice [Fig. 5(b)]. Further, the analysis of protein
extracts obtained from rye and barley seeds only
Accumulation of a wheat xylanase inhibitor protein
revealed one band, which corresponded to a
molecular mass of 30 kDa.
DISCUSSION
Measurement of xylanase inhibitor activity indicated
that xylanase inhibitor(s) were present in most parts
of the wheat plant almost throughout plant development. In contrast, the data from immunological
studies showed that XIP-I was present, at appreciable levels, only in the grains. XIP-I accumulated
during the later stages of grain development and was
still present in post-germination samples. This shows
there are distinct regulatory controls for the different
xylanase inhibitors in T. aestivum. The expression of
XIP-I to high levels in T. aestivum is restricted to the
grain and is targeted to a developmental stage, indicating that the production of XIP-I is tightly regulated. It is tempting to speculate that the xylanase
inhibitor activities observed in other parts of the
wheat plant (e.g., leaves, shoots and roots) are attributable to the TAXI type inhibitors, and that these
inhibitors are ubiquitous throughout wheat plants at
most stages of development. This hypothesis is
supported by the observation that xylanase inhibitor
activity is present at 14 days post germination, but
that no XIP-I was detected using the polyclonal
antibody. It is possible that the XIP-I independent
xylanase inhibitor activity is due to TAXI-I (and/or
other xylanase inhibitor proteins) and that while
XIP-I is degraded towards the end of germination,
other xylanase inhibitors are not.
A discrepancy between the amount of inhibitor
activity present in the latter stages of grain development and the early stages of germination was
observed, with the former being 60-fold greater
(Fig. 2). There are several possible explanations for
this observation. The large reduction in xylanases
inhibitor activity may be due to losses during dessication of the grain, a transitition of the xylanases
inhibitor proteins from predominantly soluble to
largely insoluble (e.g., as a result of being incorporated into protein bodies or becoming irreversibly
bound to cell wall polymers).
Data presented here indicates that the measurable
(1,4)-b-xylanase activity in wheat grains (germinating
or developing) is insensitive to inhibition by XIP-I or
the XIP-I-free inhibitor preparation, suggesting that
these proteins are not involved in regulation of wheat
xylanases, either post-germination or during grain
development. It was shown recently that the different (1,4)-b-xylanases identi®ed in barley12,13 represent different processing steps of a single precursor
193
protein14. The sequence of a (1,4)-b-xylanase, cDNA
from wheat15 has 911% identity to the 41 kDa
intermediate barley (1,4)-b-xylanase, and may
undergo similar processing. If so, it is unlikely that
the lack of inhibition was due to the presence of
multiple (1,4)-b-xylanases of which only some were
inhibited, and that activity detected was from (1,4)b-xylanase(s) that are insensitive to inhibition.
Inhibitor activity present in the XIP-I-free preparation may be due to TAXI-I, and indeed a band
matching the expected molecular mass (40 kDa6)
was detected following SDS-PAGE (data not presented). It has been postulated that TAXI is involved
in regulation of endogenous (1,4)-b-xylanase activity,
due to a deleterious effect observed on the addition
of TAXI to dough in the baking process6, but this
has not been supported by our results.
(1,4)-b-xylanases are active post-germination
towards the end of endosperm mobilisation in
barley12,13, wheat16 and rye17. (1,4)-b-Xylanases
have also been observed in and puri®ed from
wheat ¯our18,19 and un-germinated rye grains20.
The results presented for (1,4)-b-xylanase activity in
post-germinating grains are in accordance with those
previously reported16. However, it was shown here
that (1,4)-b-xylanase activity is also present in developing grains. The only conclusive report describing
(1,4)-b-xylanase activity outside post-germinating
grains are in regard to a (1,4)-b-xylanase present as
the predominant protein on the surface of pollen
grains in maize21. It is unlikely that xylanase activity
detected in the developing grain is due to contamination by wheat pollen (1,4)-b-xylanase, as in wheat
¯owering occurs within 3 or 4 days after head
emergence. In the experiments performed here, the
highest (1,4)-b-xylanase activity was detected 15 and
21 days after head emergence was complete. The
possibility that the (1,4)-b-xylanase activity detected
was due to microbial contamination can also be
excluded as all seed samples were surface sterilised
before protein extraction was performed. It is tempting to speculate that that (1,4)-b-xylanase activity is
present in developing grains to enable cell expansion
to occur during grain ®lling. If this were so it might
be expected that the (1,4)-b-xylanase activity would
persist throughout the grain maturation process.
However, our data indicate that the (1,4)-b-xylanase
activity decreases around 25 days post-anthesis at a
time when the grain is still maturing.
The analysis of various wheat and cereal samples
using the anti-XIP-I antibody indicate that some of
the inhibitor activity that has been observed in other
types of wheat2,22 may be attributed to XIP-I, and
194
G. O. Elliott et al.
that XIP-I is not only restricted to wheat but is also
present in rye and barley. Interestingly, no inhibitor
activity was detected in samples from rice grains.
Analysis of the primary structure of XIP-I indicated
that a putative class III chitinase from rice had a
high percentage identity (86%) with XIP-I in the
N-terminal region5. If this chitinase is present in the
grain, it would appear that the high homology was
not suf®cient for the anti-XIP-I antibody to recognise the rice chitinases, and that the chitinase did not
have xylanase inhibitor activity.
The ®nding that XIP-I does not inhibit endogenous (1,4)-b-xylanase activity but does inhibit exogenous (1,4)-b-xylanases from fungal sources, suggests
that XIP-I is involved in protecting the grain from
fungal attack during dormancy, when other plant
defence mechanisms, and in particular those that
involve an active response, are inactive or less
effective.
Acknowledgements
The authors thank Steve Reader of the John Innes
Centre, Norwich, UK for kindly donating grain samples,
and Lionel Perkins and Simon Deakin for technical
assistance.
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