Journal of Cereal Science 15 (l992) 143-149

Occurrence of Friabilin, a Low Molecular Weight

Protein Associated with Grain Softness, on Starch
Granules Isolated from Some Wheats and Related
Species
W. R. MORRISON*, P. GREENWELLt, c. N. LAWt and B. D. SULAIMAN*

*University of Strathclyde, Department of Bioscience and Biotechnology, Glasgow Gl

lSD, U.K., tFlour Milling and Baking Research Association, Chorleywood,
Hertfordshire WD35SH, U.K. and tCambridge Laboratory, Institute of Plant Science
Research, Colney Lane, Norwich NR4 7Ul, u.K.

Received 2 May 1991

Proteins of apparent M" 14-15k by SDS-PAGE were identified on starches washed

by aqueous procedures from soft endosperms of diploid Triticum and Aegilops spp.
(AA, BB, DD and SS genomes). These proteins were analogous to the previously
reported friabilins of hexaploid wheats (AABBDD genomes) and diploid rye (Secale
cereale, RR genome) and gave clear positive reactions in an ELISA test based on an
antibody to soft wheat (T. aestivum) friabilin. Little or no friabilin-like protein was
found on starches from hard endosperms of tetraploid wheats (AABB and AAGG
genomes), diploid barley (HH genome) or Aegilops umbellulata CUU genome). It is
proposed that these proteins should be designated friabilins-A, -B, -D, -S and -R,
respectively, to identify the genome carrying the controlling genes,

Introduction
Friabilin is a low molecular weight (Mr 14-15k) protein found originally on the surface
of starch granules isolated by water-washing from soft wheat (Triticum aestivum)
flours l - 3 , and there is an analogous protein on starch granules from rye (Secale
cereale)3. Examination of starches from over 300 wheat cultivars has shown an
unbroken association of comparatively high levels of friabilin with soft endosperm
texture and low levels with hard texture in T. aestivum (AABBDD genomes), and the
absence of friabilin in very hard durum wheats (T. durum, AABB genomes)1-4.
From studies with chromosome substitution lines and recombinant lines of hexaploid
wheats it has been concluded that gene(s) controlling friabilin on starch cannot be
distinguished from the major gene promoting endosperm softness 2 ,3. The latter gene,
designated Ha/ha (giving soft/hard texture) is on the short arm of the 5D chromsome 5 •
Most hexaploid triticales (AABBRR genomes) are moderately soft, whereas the
parent rye is very soft and the parent T. durum is invariably very hard a• Attribution of
both softness and the presence of friabilin to gene(s) carried on chromosome 5R was
reinforced by examination of rye chromosome addition lines, in which single rye
0733-5210/92/020143 +07 $03,00/0 © 1992 Academic Press Limited
144 W. R. MORRISON ET AL.

o b c d e h k

FIGURE 1. Electrophoregram of Coomassie-stained surface and integral proteins of

starches [150 mg, except lanes (c), (e), (I) = 90 mg] isolated from various Tl'iticeae
species by Method L Species corresponding to each lane were; (a) T. urartu (AA);
(b), (c) T. tauschii = Ae. squorrosa (DD); (d) Ae. speltoides (S8); (e), (1) Ae.
umbel/utala (UU); (g) T. dicoccoides (AABB); (h) T. dicoccum (AABB); (i) T. durum
(AABB); (j) T. lurgidum (AABB); (k) T. spetla (AABBDD), soft endosperm; (I)
T. aestillum (AABBDD), soft endosperm, cv. 'Riband '. Molecular weight markers
were used in end lanes. Arrows indicate principal integral protein at MI' = 59k and
friabilin region at M r = 15k.

chromosome pairs from King II rye had been added to the 21 chromosome pairs of the
hard wheat variety Holdfast. Only addition of chromosome 5R caused the Holdfast to
exhibit soft texture and a high level of friabilin (the rye analogue) on the starch 3 .
Thus, high levels of rye friabilin are also associated with soft endosperm texture in rye
and triticales, although it should be noted that 280 triticale cultivars tested at ICARDA,
Syria, exhibited the complete range of hardness (measured by particle size index) found
in T. durum and T. aestivum cultivars 6 • Hence, there could be as much variation in levels
of rye friabilin as there is in hexaploid wheat friabilin, but this is not certain because
elements of the R genome may have been displaced by the D genome in natural or
deliberate out-crossing of the primary triticale with hexaploid wheat to produce
secondary triticales.
We developed a method recently for isolating pure starch from single kernels of wheat
and similar grains that depends on centrifugation through 80 % (v Iv) caesium chloride
to purify the starch 7. The process removes most of the surface proteins, except for
friabilin, which is recovered nearly quantitatively on the surface of the starch 8 • The
method is therefore suitable for screening small samples of grain and starch for friabilin.
This paper describes a survey of diploid and tetraploid Triticeae species for the
occurrence of friabilin-like proteins, as the first part of a study of the genetic control of
friabilin in wheat and related species.
 FRIABILIN IN TRITICEAE 145

Experimental
All Triticum, Aegilops and Secale samples were from stocks held at the Cambridge Laboratory,
Institute of Plant Science Research, Norwich and Hordeum samples were from the Scottish Crop
Research Institute, Dundee. Starch was isolated for analysis of granule surface proteins in two
ways.

Method 1
Grain (c. 600 mg) was reduced to flour in a coffee grinder and replicate samples (250 mg) weighed
into tubes. If normal white flour was available the sample size was reduced to 200 mg. Flour
samples were defatted once with light petroleum (b.p. 40-60 °C; I ml) and twice with acetone
(I ml). The defatted flour was then homogenized (Potter homogenizer, clearance 0·1 mm) with
water (3 ml) four times and the combined starch slurries flushed with a pipettor through a woven
nylon screen (10 l!m nominal aperture, but flexible, which removed gelatinous cell wall flakes while
allowing passage of oversize A-type starch granules - confirmed by microscopy and Coulter
particle size analysis; A. D. Evers, pers. comm.). Crude starch was recovered by centrifuging at
2000 g for 15 min. The pellet was then taken up in water (10 ml), and 80% (wjv) CsCI (2 ml)
introduced carefully as a discrete layer at the bottom of the tube, which was then centrifuged at
3000 g for 20 min. The starch pellet was retained and the supernatant layers of CsCl solution,
proteinaceous floating debris and water discarded. The starch pellet was centrifuged through CsCI
a second time, then washed with water (3 x 5 ml), acetone (5 ml) and air-dried.
Surface and integral proteins were extracted from starch (c. 150 mg) with 2% (wjv) sodium
dodecylsulphate (SDS; 2 ml) at 50°C for 30 min for electrophoresis on polyacrylamide gradient
gels (SDS-PAGE, 7·5 to 25% polyacrylamide) as described previouslyl. Gels were stained with
Coomassie Blue R250.

Method 2
Starch was isolated from cracked whole grain by steeping, grinding gently and centrifuging
through 80 % (w jv) caesium chlorides. Surface proteins, together with the majority of the integral
proteins, were extracted from samples of purified starch (c. 100 mg) with 2 % (wjv) sodium
dodecylsulphate (SDS) at 50°C, and separated by SDS-PAGE (10-20 % polyacrylamide) and
silver stained as described previously8.
In both methods the weight of the swollen starch (typically 0·9-1'1 g when using 150 mg dry
starch) was noted, and if it indicated swelling within the normal range it was assumed that
satisfactory yields of the main integral protein and near quantitative yields offriabilin and other
surface proteins had been obtained (in practice no anomalies were found). The principal integral
protein (M,. 59k) served as an internal standard. The characteristic band at M,. 14-15k was
assumed to be friabilin, and band intensities were scored subjectively on a scale of 0 to 10 in those
lanes where the M" 59k bands were of uniform intensity.
Friabilin was also quantified using an enzyme-linked immunosorbent (ELISA) assay (W. H.
Stimson and P. Greenwell, in preparation), exhibiting high specificity for friabilin from hexaploid
wheat (T. aestivum, AABBDD genomes) and negligible reaction to other endosperm proteins.
Since the method was not calibrated the results cannot be given in absolute terms, but they were
nevertheless unequivocally positive or negative.

Results and Discussion

Electrophoregrams of all starches showed similar band patterns for integral proteins
(M,. > 30k), including a strong band for the major component (M r 59k), which has been
tentatively identified as granule-bound starch (amylose) synthase 1 • 2. 9. Since surface
146 W. R. MORRISON ET AL.

- 59k

-15k

FIGURE 2. Electrophoregram of silver-stained surface and integral proteins of

starches (100 mg) isolated from various Triticeae species by Method 2. Species
corresponding to each lane were: (a) T. monococcum (AA); (b) T. tauschii = Ae.
squarrosa (DD); (c) T. carthlicum (AABB); (d) T. dicoccoides (AABB); (e) T. durum
(AABB); (f) T. turgidum (AABB); (g) T. timopheevi (AAGG); (h) T. aestivum
(AABBDD), soft endosperm, cv. Brigand.

proteins on cereal starches are extracted much more readily than the integral proteins 2 • 8,
it was concluded that recoveries of the surface proteins were quantitative. Figures I and
2 show typical electrophoregrams.
Although esCI treatment is very effective for removing surface proteins other than
friabilin from the starches of hard and soft common wheats 7 • 8 , it was not always so
efficient with starches from some other species studied here. The latter group had
variable amounts of other surface proteins that were not removed even by a double
treatment with caesium chloride, but they did not obscure (or compare in intensity with)
the friabilin bands. While many barley starches had appreciable quantities of these other
surface proteins, in the M r 15k region of the gels, there was either a faint band in some
cases only (starch prepared by Method I) or no discernible friabilin analogue at all
(Method 2).
 FRIABILIN IN TRITICEAE 147

TABLE 1. Occurrence of friabilin in wheats and related species

Friabilin Friabilin
Species Genome(s) score' Species Genome(s) score

T. monoeoeeum AA 10** T. carthlieum AABB Orl

T. urartu AA 10 T. dicoecum AABB Od
Ae. bieomis 10** T. dieoecoides AABB 0
Ae. longissima
Ae. mutiea
SIS'
S"S"
M'M'
J 10*
10*
T. durum"
T. turgidium
AABB
AABB
Od
Od
BBb
Ae. sears if S'S' 10*** T. timopheevi AAGG Od
Ae. sharonensis SIS' 10** T. aestivum a AABBDD 1-6*1**
Ae. speltoides SS 10* T. spelta AABBDD 6-10
Ae. squarrosa DD 10* Triticale a AABBRR 2-6*
(T. tauschi)
Ae. umbel/ulata UU 0-1
H. vulgare HH 0
(2-row, 6-row)
S. cerea/e a RR 10

a Includes results described in the literature H .

b The genomes of this group of diploids are closely related and it is considered that the Band G genomes
originated from within this grouplO.
, Asterisks indicate strength of ELISA positive reaction.
d Negative.

Table I shows the relative intensities of friabilin bands on starches isolated from
diploid, tetraploid and hexaploid species related to common wheat. Results for rye (s.
cereale), triticale and common wheat (T. aestivum) include results from water-washed
starches described by us in the literature1-3 to give a comprehensive picture. With the
exeption of H. vulgare (two-row and six-row barleys) and Ae. umbellulata, which had
little or no traces of friabilin, all diploid species gave very strong bands in the MI'
14-15k region, which were operationally defined as friabilins by analogy with common
wheat friabilin. The relative intensities offriabilin bands scored from electrophoregrams
(Table I) were unequivocally confirmed by ELISA results for all diploid and tetraploid
species that were tested, and were not affected by the presence of other surface proteins
on starches from H. vulgare and some of the tetraploid species.
In common wheat 2 there is a controlling gene for the level of friabilin on starch on
chromosome 5D, and in rye 3 the corresponding gene is apparently on chromosome 5R.
The' controlling gene' could be the friabilin structural gene, or the gene for an unknown
product that controls either the expression of the friabilin gene or the adhesion of
friabilin to the starch granules. The results on diploids presented here show that there
must also be controlling genes for friabilin in the A genome and in those related to the
Band G genomes ofpolyploids 1o , probably on the homoeologous group 5 chromosomes.
The controlling gene in Ae. squarrosa (D genome) may be the gene expressed in
hexaploid wheat.
Although they may have evolved from a cornman ancestor it is unlikely that the alleles
of the friabilin genes in the diploid species representing the A, B, D and R genomes
148 W. R. MORRISON ET AL.

(Table I) are identical, and, therefore their friabilins may not be identical. We suggest
that these friabilins should be designated friabilin-A, friabilin-B, friabilin-O and
friabilin-R to identify the genome carrying their controlling gene(s). Friabilin-R has a
slightly lower apparent M r value than friabilin-O of hexaploid wheat 3 • Many of the
friabilins appeared to consist of two poorly resolved components, and we have other
evidence (PG, unpublished results) that friabilin-D consists of a basic cysteine-rich
polypeptide and perhaps two minor near-neutral polypeptides. More sophisticated
structural analysis than 80S-PAGE should reveal whether friabilins A, Band Dare
different, or not.
H is remarkable that no friabilins were detected in any of the tetraploid species
because representatives of the probable diploid ancestors all gave strong friabilin bands.
This suggests that the friabilin-A, -B and -G structural genes may have been null in the
tetraploids, or their expression was totally blocked by unknown trans-acting genetic
elements. Variation in the regulation of expression of genes for friabilins-D and -R is
indicated by the range of scores for T. aestivum and triticale (Table I), although in the
case of the triticales this conclusion is qualified by uncertainty about the completeness
of the R genomes. Another possibility that cannot be excluded is that the controlling
genes for friabilins-A and -B were active in a few of the T. aestivum and triticale
cultivars.
For an experienced operator it is not difficult to distinguish between individual cereal
grains with hard and soft endosperm texture when isolating starch by either method used
here, and it is even possible to rank T. aestivum cultivars and group 5 aneuploids for
hardness/softness with reasonable confidence. All the tetraploid species and H. vulgare
were judged to be very hard, while all the other diploids (AA, BB or DO genomes) except
Ae. umbellulata were extremely soft, as reported previously6. There was insufficient
material to do quantitative objective measurements of hardness for the majority of the
species studied.
Thus, the association between friabilin band intensity and grain softness in over 300
lines of wheat and in wheat/rye chromosome addition lines l - 3 (with some possible
exceptions 4 ) now extends to all species of the subtribe Triticineae for which studies have
so far been completed.
Whether the association extends to other branches of the Triticeae tribe such as
Hordeum species is not completely clear. Both two- and six-row barleys are known
to exhibit variation in endosperm texture. Indeed, soft ('mealy') endosperm is much
preferred to hard (' steely') endosperm for malting quality barley, but this quality has
been assessed by a 'milling energy' test l l that differs greatly from the indices of flour
particle size ofwheat 3 • 6that have been used to interpret the present work. Thus, the zero
or low levels of a putative' friabilin- H ' seen in the present work may not be incompatible
with the variation in barley endosperm texture. In milling energy terms, most barleys are
harder than most breadwheats (R. P. Ellis, W. R. Morrison and P. Greenwell, un-
published results), and the difference between hard and soft barleys might therefore be
more akin to that between durum wheats and hard breadwheats. Clearly, more work is
required to clarify the relationship between friabilins and endosperm texture for both
Hordeum and Triticum species.
 FRIABILIN IN TRITICEAE 149

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