He edi as 132: 1 1 1
-
1
18
(2000)
The mos abili ies
o
g ain p-amylase and p-glucanase in Finnish
land ace ba leys and hei pu a i e pas adap edness
HANNU AHOKAS and MARJA-LEENA MANNINEN
C ops and Soil, Ag icul u al Resea ch Cen e, Jokioinen, Finland
Ahokas,
H.
and Manninen, M.-L.
2000.
The mos abili ies o g ain (3-amylase and (3-glucanase
in
Finnish land ace ba leys
and hei pu a i e pas adap ednes-He edi as
132:
11 1- 118. Lund, Sweden. ISSN 0018-0661. Recei ed No embe 19,
1999. Accep ed Ma ch
6,
2000
The mos abili y o (3-amylase ac i i y was a gene al ea u e in a sample o 32 Finnish ba ley land aces. One
o
wo
Finnish land aces p obably con ibu ed he he mos abili y o c . 'Pi kka' in c osses pe o med abou 70 yea s ago. The
s abili y is less e ol ed in P-glucanase ac i i y al hough he mos ole an ypes appea ed in land aces and in Pi kka wi h
a
Finnish land ace backg ound. Selec ion'p essu e o he mos abili y
in
g ains may ha e been a ea u e o adi ional
c op managemen p ac ices among Finns in he pas : d ying g ain c ops, including p ema u e ba ley, abo e an o en in
a special d ying house a empe a u es exceeding
55"C,
and ge mina ion in black, sunli slash-and-bu n soils, wi h a
measu ed su ace empe a u e o
63°C.
A posi i e, hough small co ela ion be ween he he mo ole ance a ios
o
he wo
enzymes may be
a
emnan
o
hei common long selec ion p essu e ending ens o gene a ions p io o collec ion in he
1960s and 1970s.
Hunnu
Ahokus,
C ops
and
Soil,
Ag icul u al Resea ch Cen e,
Mylly ie
10,
FIN-31600
Jokioinen,
Finland. E-mail:
[email p o ec ed]
Samples o Finnish land ace ce eals s udied p e i-
ously p o ed highly a iable in se e al ai s, e i-
den ly con aining a ich a ie y o genes and
combina ions o genes. The land ace popula ions
ha e been mix u es, some app oaching mix u es
o
unique geno ypes in he pas (AHOKAS 1998;
AHOKAS and POUKKULA 1999). The easons o his
a ia ion a e e iden ly his o ical, en i onmen al and
selec i e (AHOKAS and MANNINEN 2000). Me hods
o c op and ield managemen p io o abou 1930 in
Finland may ha e gene a ed en i onmen s which un-
consciously selec ed he mos abili y o a ious i al
cha ac e is ics a ge mina ion and ha es . The he -
mos abili y o enzymes o mal ing ba ley (Ho deum
uulga e L.) is gene ally a desi ed cha ac e is ic, and
p e e ed mal ing ba leys appa en ly ha e signi ican
P-amylase he mos abili y (KIHARA e al. 1998). They
also
show pedig ees wi h simple inhe i ance o he -
mos abili y (KIHARA e al. 1998). Since high he -
mos abili y o P-amylase exceeding 65
YO
emaining
ac i i y seems o be a a e bu an inhe i ed cha ac e -
is ic (KIHARA e
al.
1998, 1999), he e is eason o
s udy he mos abili y in he sample, p obably man-
aged wi h slash-and-bu n cul u e (see HEIKINHEIMO
191
5)
and iihi-hea ed d ying (see GROTENFELT 1899;
TALVE
1961) s ill some ens o gene a ions ea lie .
MATERIAL AND METHODS
Plan ma e ial
The ma e ial analyzed in his s udy, which has been
desc ibed ea lie (AHOKAS and POUKKULA 1999),
was om he 1996 ha es and o good quali y. Mos
o he Finnish land ace selec ions ha e been acces-
sioned by he
US
Depa men
o
Ag icul u e,
Bel s ille, MD: PI 349678-PI 349681, PI 415017-PI
415019, PI 467622-PI 467627 and PI 467629-PI
467653.
Sound g ains we e weighed and hulled pa ly by
hand and u he wi h a
50%
H,SO,
wash ollowed
by wa e inses, and hen ge mina ed asep ically on
washed, s e ile qua z sand in g oups o
15
in he
da k a 15.5
0.5"C o 120 hou s. The ea e , he
ge minan s we e homogenised asep ically in
a
bu e
o
pH 4.6 con aining 40 mM sodium ace a e, 40 mM
sodium phospha e and 0.001
%
sodium azide as p e-
iously desc ibed (AHOKAS and POUKKULA 1999).
The ex ac supe na an s we e s o ed a -70°C o
empo a ily a -20°C un il used. The assays we e
eplica ed and eplica es which di e ed by mo e han
ou pe cen age poin s we e analyzed o a hi d ime.
All he esul s a e gi en as means
o
he
de e mina ions.
112
H.
Ahokas and
M.-L.
Manninen
He edi as
132
(2000)
Assay
o
@-amylase
Aliquo s
(40
pl)
o he ex ac we e mixed wi h 3960
pl o a cold bu e
o
50
mM MOPS,
pH 7.0 wi h 1
%
BSA (Sigma A-751 1). A sample o
200
p1
was kep on
ice and ano he hea ed o 30 min a 56.7"C in a
he mos a ic ci cula o (LKB 2219 Mul i emp
I1
The mos a ic Ci cula o using
20
YO
Shell An i eeze
402
coolan in he wa e ba h). The ac ual empe a-
u e anged
om
56.3 o 57.0"C du ing he incuba-
ion as measu ed by he ins umen and an ex e nal
he mocouple (P 1000, Knick). Samples o 25 pl o
he hea ed and +0"C con ol samples we e u he
dilu ed wi h 225 p1 o bu e B
(100
mM maleic acid,
1
mM EDTA,
0.1%
w/
o
BSA,
NaOH un il pH 6.2
was eached and 0.02% sodium azide) and assayed
wi h a
p
-ni ophenyl mal open aoside subs a e con-
aining a-glucosidase pu chased om Megazyme.
Table
1.
p-Amylase ac i i y in ex ac s
o
ge mina ed g ains a e
30
min incuba ion a
56.7"C
Land ace o e e ence Remaining ac i i y ( a io) Ac i i y
wi hou
incuba ion a
56.7"C
(a bi a y uni s o g ain mass)
Land ace
selec ions
HA
22
HA
44
HA
52
HA
9
HA
20
HA
10
HA
38
HA
31
HA
42
HA
53
HA
17
HA
29
HA
19
HA
49
HA
18
HA
40
HA
9-63-4
HA
70-3
HA
6-33-02
HA
9-63-8
HA
70-2
HA
5
HA
48
HA
11
HA
12
HA
33
HA
9-63-2
HA
9-63-1
HA
3
HA
14
HA
45
Global ba leys
Ha una Nijo
Pi kka
Noi e
2R
Mon pellie
PI
391421
Ado a
F(6,
lines
a
HA
52
x
Ado a
HA
52
x
Ado a
HA
52
x
Ado a
HA 52xAdo a
HA
52
x
Ado a
HA
146-04-1
0.90
0.82
0.77
0.77
0.76
0.76
0.75
0.75
0.75
0.74
0.74
0.74
0.74
0.73
0.73
0.73
0.73
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.71
0.71
0.71
0.70
0.41
0.39
0.87
0.75
0.40
0.38
0.38
0.65
0.44
0.41
0.40
0.40
0.62
0.74
0.69
1.63
0.86
0.49
1.05
1.13
0.78
1.01
0.54
0.47
0.51
0.98
0.68
1.12
0.77
1.03
0.64
0.65
0.48
0.89
0.52
0.98
0.81
1.08
0.81
0.61
0.89
0.76
0.24
0.40
0.66
0.68
0.36
0.36
0.57
0.34
0.36
0.30
0.88
0.61
a
G ains
om
se e al
F,
plan s.
He edi as
132
(2000)
Enzyme he mos abili y
in
ba ley land aces
1
13
Table 2.
Spea man coe jcien s
o
ank co elu ion
be ween a ios
o
p-glucanase he mos abili y
and
o he independen measu emen s in he
32
ba ley
lan-
d ace samples
Second a iable s Signi icance
Ac i i y
o
P-glucanase wi hou
0.140
NS
(P
=
0.56)
hea
ea men
( o
g ain
mass)
hea
ea men
( o
ex ac
olume)
Ac i i y o b-glucanase wi hou
0.058
NS
(P
=
0.75)
The mos abili y o P-amylase
0.277
P
=
0.12
The mos abili y
o
P-amylase
0.351
P
=
0.057
excluding
wo
ex eme
a i-
an s
(Fig.
2)
'
Wi hou hea ea men , ac i i y
o
P-glucanase o g ain
mass
s
ex ac olume,
s
=
0.944,
P
<
0.001.
The assay was conduc ed acco ding o he supplie 's
ins uc ions and ook 10 niin a 40°C. The dilu ions o
he ex ac s we e 1000- old o he assay, dilu ing
pu a i e endogenous he mop o ec ing molecules, e.g.
mal ose (TAKAHATA e al. 1994), and enzyme in-
hibi o s o insigni ican le els.
Assay
o
B
-glucanase
Mel ed and well-mixed ex ac s we e dilu ed 3.76- old
wi h Na-ace a e bu e (25 mM, 0.02Y0 wj o Na-
azide, inal pH 4.43) and
1
YO
w/ o BSA (Sigma
A-751 1). Samples o
550
pl we e ei he hea - ea ed o
15 min a
+
45.0"C as desc ibed o kep on ice. Bo h
he samples we e le o s and o 30 min a oom
empe a u e, wi h subsequen assaying o
500
p1 a
+30°C o 15 nin wi h a Be a-Glucazyme able
(Megazyme) based on Azu ine-c osslinked ba ley
P-
glucan. The eac ion was e mina ed wi h 6 ml o
1
YO
w/ T izma base in wa e , o exed wice a
5
min
in e als, il e ed (Wha man
1,
0
9 cm) and ab-
so bances we e measu ed a 590 nm as ins uc ed by
he supplie (Megazyme). The inal assay pH was 4.8
a 30"C, and was main ained du ing he hea ea men .
In Na-ace a e bu e he maximal ac i i y has been
obse ed a pH
5
(KOTAKE e al. 1997). Abso bances
we e de e mined in a bi a y uni s based on he ex ac
olume o he o iginal g ain mass.
RESULTS
p-amylase
The ac i i y emaining a e hea ing is p esen ed in
Table
1
as he a io
o
32 land ace seIec ions, i e
global ba leys and i e lines o he c oss HA
52
x
'Ado a'. Among he global ba leys, 'Ha una Nijo',
known o ha e he mos able P-amylase based on
ex ac s o unge mina ed g ains (KIHARA e al. 1998),
appea ed o ha e he mos able P-amylase in his s udy
o ge mina ed samples (Table 1). 'Pi kka' (also s udied
as a4459), known o ha e highly ac i e P-amylase
(SIMBERG 1950; ALLISON and
SWANSTON
1974),
p o ed o ha e highly he mos able P-amylase in his
s udy, while he o he global ba leys ha e he lowes
a ios, wi h le els anging om 0.38 o 0.40. Fi y pe
cen o he pa en age o Pi kka is om wo Finnish
land aces (SIMBERG 1950;
KIVI
1969), he Speci ic
pa en al lines o land aces c ossed abou 70 yea s ago
no being main ained. The a io dis ibu ion o he
land ace samples a ies om 0.38 o 0.90 wi h a
mean SEM o 0.72
0.09, hei o al dis ibu ion
de ia ing highly signi ican ly om no mali y
(x'
=
511,
P
<<
0.001). The cen al ac ion, land aces wi h
he wo highes and wo lowes a ios emo ed, anges
om 0.70 o 0.77 wi h a mean SEM o 0.73
0.003,
and i s a no mal dis ibu ion
(x2
=
2.154, P
>
0.80).
This sugges s ha he ange o 0.70 o 0.77 is p oduced
by
a
single allele o se e al allele ypes ha ing he same
e ec . The e seem o be o he alleles in ol ed, pu a-
i ely one causing 0.39 o 0.41 a ios, and wo o he s,
one gi ing a a io o 0.81 and he o he gi ing 0.90
(Table 1).
The e is no co ela ion be ween he a io o he -
mos abili y and he o al unhea ed P-amylase ac i i y
in a gi en olume o he sample ( s
=
0.196, P
>
0.30),
o be ween he a io and he ac i i y pe uni o g ain
mass in he sample ( s
=
0.176, P
>
0.40).
p
-Glucanuse
The ac i i y
o
P-glucanase is in gene al less he -
mos able han ha
o
P-amylase. The ac i i y o he
non-hea - ea ed ex ac s a ied om 0.51 o 1.29
a bi a y uni s pe ml in he samples o he 32 land aces
and om 0.75 o
1.21
a bi a y uni s in he samples o
he i e global ba leys. Rela i e o g ain mass, he
a ia ions in ac i i y we e 0.80 o 2.24 a bi a y uni s
in he 32 land ace samples and 1.05 o 1.86 a bi a y
uni s in he i e global ba ley samples. The co ela ion
o
hese ac i i y de e mina ions
o
he 32 land aces was
s =0.944 (P
<
O.OOl), and ha
o
he i e global
ba leys s
=
0.60
(NS)
(Table 2).
The a ios
o
he ac i i y o he emaining hea -
ea ed
(15
nin a 45°C) P-glucanase o he o iginal
ac i i y a ied om 0.52 o 0.81 wi h a mean o 0.67
in he 32 land aces, om 0.40 o 0.82 wi h a mean o
0.59 in he i e global ba leys. Among he global
ba leys, Pi kka, wi h a
50%
Finnish land ace back-
g ound, has he highes emaining ac i i y a io o 0.82,
he o he a ios being
0.71
(Ha una Nijo), 0.59
(Ado a), 0.45 (Noi e
2R
Mon pellie ) and 0.40 (PI
391421).
114
H.
Ahokus
and M.-L. Munninen
He edi as
132
(2000)
The dis ibu ion o he mos abili y in he land ace
sample (Fig. 1) de ia es om no mali y due o la ness
and is bimodal wi h cen al modes o 0.61 and 0.73.
Va ious co ela ions a e p esen ed in Table 2: he e is
a small posi i e co ela ion be ween P-amylase and
P-glucanase he mos abili ies, s
=
0.277, P
=
0.12, and
i wo ex eme a ian s a e exluded, s
=
0.351, P
=
0.057 (Fig. 2).
DISCUSSION
-amylase
High he mos abili y o P-amylase in ba ley cul i a s
appea ed a e and displayed inhe i ance in he known
pedig ees (KIHARA e al. 1998). In hese land aces, he
o al P-amylase ac i i y o g ain mass o soluble
p o ein is highly a iable (AHOKAS and POUKKULA
1999). The lack
o
co ela ion indica es ha ac i i y
le el and he mos abili y a e sepa a e phenomena and
p obably ha e a di e en gene ic basis.
The a ios o he i e
F(6)
lines
o
he c oss HA
52
x
Ado a, pa en s wi h 0.77 and
0.38
a ios, e-
spec i ely (Table
l),
indica e ha he he mos abili y
a io has a simple inhe i ance as shown by o he
ma e ial
(KIHARA
e al. 1998). The line wi h a 0.65
a io may s ill ha e a he e ogeneous mino i y o
g ains wi h he low- a io allele. Allelic di e ences in
he inal amino acyl sequence P-amylases ha e been
de ec ed in ba ley
(KREIS
e al. 1987; ERKKILA e al.
1998) o induced in cloned ba ley sequencies
(OKADA e
al.
1995). Amino acyl esidue changes
ha e been ound o con e he mos abili y
(OKADA
e al. 1995; EGLINTON e al. 1998; MIKAMI e al.
P-Amylases as p o eins appea o be mul i unc-
ional in a ious plan species and hei di e en
issues (PAN e al. 1988; AHOKAS and NASKALI
1990; GANA e al. 1998), he enzyme ac i i y no
necessa ily being he objec i e o na u al selec ion
e.g. unde he mos ess.
1999).
REMAINING ACTIVITY
(%I
50
55
60
65
70
75
80
4
%
ic
33
a
Y
0
12
=
E
1
0
g
0.25
i?
6
0.50
3:
0.75
2
1.00
1.25
1.50
)
c
I-
F
U
U
-I
-
u_
~-
I
1
I
I
I
I
II
I'
Fig.
1.
The bimodal dis ibu ion
o
he emaining ac i i y
o
P-glucanase pe cen -
ages among he
32
land ace lines (uppe plo ), and he o iginal un ea ed ac i i ies
(lowe plo ).
He edi as
132
(2000)
Enzyme he mos abili in ba ley land aces
115
em
Ip
**
**
**
**
*.*
I
1
1
I
I
I
40
50
60
70
80 90
B-AMYLASE, REMAIIUING ACTIVITY
(%)
Fig. 2.
The
dis ibu ions
o
B-amylase and /3-glucanase he mos abili ies in he
32
land ace lines
sugges ing
an in luence
o
pas coadap a ion. Spea man coe icien s
o
ank co ela ion, s
=
0.277,
P
=
0.12 o he whole sample;
i
he wo ex eme
a ian s o he le a e excluded,
s
=
0.351,
P
=
0.057.
-
Glucanase
The ac i i y o unhea ed P-glucanase showed less
a ia ion han be a-amylase o a-amylase
(AHOKAS
and POUKKULA 1999). This holds ue o a wild
ba ley sample o 257 H.
spon aneum
en ies (AHOKAS
and POUKKULA 1999). Since P-glucanase loosens cel-
lula walls, inc easing pe mea ion (H~J and
FINCHER
1995), i s excessi e ac i i y esul s in he dange
o
leakages om he ge mina ing g ain may also be a
disad an age. While
1
+
3-P-glucanase has pa hogen-
esis- ela ed e ec s agains ungi, inc easing hei cell-
wall pe meabili y (see e.g.
GRENIER
e al. 1999), he
1
-+
3,l
-+
4-P-glucanase makes he cell walls o he
we ed and ge mina ing g ain issue mo e suscep ible
o in ading o ganisms.
Up o
5
QTLs
o inished mal glucanase and
3
QTLs o g een mal glucanase we e de ec ed (HAN e
al. 1995) wi h wo s uc u al genes o isoenzymes
o
(1
+
3,1+
4)-P-glucanase (LITTS e al. 1990; WOLF
1992). One
o
hese, EII, is es ic ed o he aleu one
laye
o
ge mina ed g ain, while
EI
is also ansc ibed
in scu alla on young lea es and oo s a ge mina ion
in addi ion o aleu one (SLAKESKI e al. 1990;
SLAKESKI and
FINCHER
1992). The P-glucanase
isoenzyme I1 was ound o be glycosyla ed wi h 3.6%
ca bohyd a e
(WOODWARD
and
FINCHER
1982).
Glycosyla ion may be he sou ce
o
he mos abili y in
bac e ial P-glucanase (OLSEN and
THOMSEN
199 1)
and many o he ypes
o
p o eins (e.g.
Gu
e al. 1989;
NAKAMURA e al. 1998;
YANEz
e al. 1998). The
le el o glycosyla ion is possibly subjec o mul igenic
a ia ion.
The obse ed bimodali y sugges s wo alleles, pe -
haps wo ypes o glycosyla ion EII gene p oduc ,
and may also mean a mo e complica ed dependence
wi h wo le els
o
EI ac i i y masking he EII ac i i y
le els. The unhea ed and hea ed ac i i y did no show
any co ela ion (Table 2), sugges ing ha he mos a-
bili y is independen
o
ac i i y in his land ace sam-
ple. The high he mos abili y ound in Pi kka, wi h a
50%
land ace pa en age (SIMBERG 1950; KIVI 1969),
p obably has i s o igins in Finnish land aces.
This small sample does no necessa ily e eal ei he
he ex eme ac i i ies o he he mos abili y
o
he
pas a ia ion in he Finnish land aces. A gene ically
modi ied bac e ial P-glucanase has high he mal s a-

116
H.
Ahokas and
M.-L.
Manninen
He edi as
132
(2000)
bili y (JENSEN e
al.
1996, 1998). T ansgenic ba ley
exp essing bac e ial P-glucanase has shown s abili y
o he gene o e a ew gene a ions (JENSEN e al.
1998) and may hence se e as an a i icial al e na i e
o he endogeneous esou ce in ba ley, al hough lan-
d aces ha e no ye been h oughly sc eened.
Gene al discussion
Selec ion o s able p o ein o ms by epea ed ex e nal
hea may ha e occu ed in he land aces. The e a e
wo s ages du ing which Finnish land ace ce eals
we e o en subjec ed o hea in he pas . The ha -
es ed ma u e and p ema u e s aws we e equen ly
d ied o e a special o en called a
kiuas,
gi ing o
pe usi e smoke in
a
special building called
a
iihi
(TALVE 1961). Du ing such d ying, c ops we e com-
monly subjec ed o ini ial empe a u es o 55-60°C;
he empe a u es we e la e aised, and excessi e
hea ing some imes occu ed
(GROTENFELT
1899,
1922). Viable g ain issues we e some imes subjec ed
o dena u a ing hea .
The o he s age a which g ains may ha e been
subjec ed o ex a hea ing occu ed a ge mina ion in
black slash-and-bu n soils. Di e en a ian s
o
bu ning as
a
mode o cul i a ion (e.g. bu n o e
pea land) did no end in Finland un il he 1940s
(AHOKAS and MANNINEN 2000) and bu ning was he
p e ailing me hod o ield managemen in he pas
(HEIKINHEIMO 1915). A da k soil su ace, such as
ha o
a
bu ned a ea, abso bs mo e sola adia ion
and hus becomes ela i ely ho . Du ing di e en
summe s in Finland a N la i udes
o
61'40' and
61 '52', espec i ely, he maximum empe a u es mea-
su ed in he su ace laye o bu ned black soil has
been 52.8"C (LIPAS and MAKI-PE AYs 1961; VIRO
1974), and up o 63°C on he soil su ace
(VAARTAJA
1949). In he pas , ba ley commonly ge mina ed in
ea ly June, and hence he bu ned soils se ed as
a
hea -selec i e agen due o hei da kness. Soil em-
pe a u e maxima exceeding 50°C would be excep-
ional du ing he ge mina ion season e en in he
sub opical dese habi a s
o
wild ba ley, bu a e
eached o ba ley
a
he seasonal end o pos -season-
ally (GUTTERMAN 1997). Wild ba ley has also been
a
sou ce o he mos abili y in g ain P-amylase
(EGLIN-
TON
e
al.
1998; AHOKAS and NASKALI, unpub-
lished). Due o he appa en mul i unc ion
o
ba ley
P-amylase, o he easons o he enzyme he mos a-
bili y canno be excluded.
The ba ley enzyme P-glucanase is induced a ge mi-
na ion (e.g. BRUNSWICK e al. 1987; SLAKESKI and
FINCHER
1992). The e o e, slash-and-bu n manage-
men pu a i ely p o ided
a
he moselec i e en i on-
men o ba ley.
The
1
+
3,l +4+-glucanase isoenzymes
EI
and
EII
a e he p inciple ac i i ies expec ed o appea in
samples ge mina ed o i e days (BRUNSWICK e al.
1987;
LOI
e al. 1987; MCFADDEN e
a].
1988), wi h
subs a e speci ici y owa ds mixed-linked 1
-+
3,l
-+
4-P-glucans
(HBJ
and
FINCHER
1995). Mal ed ba ley
(1 -3,l +4)-P-glucanases we e ound o be he mo-
labile (BRUNSWICK e al. 1987). The signi icance o
b ewing is indica ed by he ac ha he ac i i y o
P-glucanase du ing mal ing is posi i ely co ela ed
wi h mal ex ac (STUART e al. 1988).
The
a e
o
ce eal land aces
I has u ned ou
o
be a subs an ial
loss
o local
plan b eede s ha he Finnish land aces, hemsel es
a pa o he na ional he i age, ha e no been ade-
qua ely main ained. The gene ical mix u es
o
lan-
d aces o sel -pollina ed ce eals we e los in abou 50
yea s p io o 1955 (AHOKAS 2000). The endange ed
s a e o he na ional land aces was poin ed ou by
PESOLA (1951) a e he opic was discussed a he
8 h In e na ional Gene ic Cong ess in S ockholm
(KIRK 1949), bu he u gen collec ing and main-
ainance p oposed by
PESOLA
(1951) emained
un ealized.
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