He edi as 134: 91-95 (2001)
~~~
B ie epo
Special ba ley P-amylase allele in a Finnish land ace line
HA52
wi h
high g ain enzyme ac i i y
MARIA
J.
ERKKILA and HANNU AHOKAS
Plan P oduc ion Resea ch, Ag icul u al Resea ch Cen e, Mylly ie
10,
FIN-31
600
Jokioinen, Finland
E-mail: [email p o ec ed]
(Recei ed Feb ua y 19, 200 1. Accep ed May 2 1, 200 1)
Ba ley
(Ho deum uulga e
L.) g ain mainly consis s o
s a ch, which p o ides ene gy du ing ge mina ion
and seedling g ow h ( o e iew, see MACGREGOR
and
FINCHER
1993). S a ch deg ada ion equi es con-
ce ed ac ion o limi dex inase, P-amylase, a-glu-
cosidase (SUN and HENSON 1990) and a-amylase.
Libe a ion o mal ose and limi dex ins om he
non educing ends o s a ch is ca alysed by P-amylase
(1,Ca-D-glucan mal ohyd olase, EC 3.2.1.2) (ROBYT
and WHELAN 1968; SOPANEN and LAURIERE 1989).
Being syn hesised du ing g ain de elopmen (KREIS
e al. 1987), P-amylase is one o he majo p o eins
ound in he s a chy endospe m (HEJGAARD and
BOISEN 1980). The endospe m P-amylase gene
(P-
amyl)
is loca ed in ch omosome 4H (KREIS e al.
1987). Ano he P-amylase gene
(P-amy2),
called ubiq-
ui ous, is loca ed in ch omosome 2H and he p o ein
is ound in lea es and oo s (KREIS e al. 1988;
SHARP e al. 1988).
Mode n plan b eeding has educed he gene ic
a iabili y in domes ica ed ba ley (THOMPSON e al.
1990;
FORSTER
e al. 1991) and declined land aces in
Finland p io o abou 1950 (AHOKAS 2000). La ge
gene ic di e si y is ound in wild ba ley
H. ulga e
ssp.
spon aneum
(K. Koch) A.
&
G ., abb e ia ed as
H. spon aneum
(AHOKAS 1982; ZHANG e al. 1993;
SAGHAI MAROOF e al. 1995). High P-amylase ai
was inhe i ed in he backc ossed p ogeny o
H. spon-
aneum
and domes ica ed ba ley (AHOKAS and
ERKKILA 1992). Howe e ,
H. spon aneum
was no
widely used in b eeding o se e al easons such as
sha e ing o spikele s, ad e se g ow h y hm a high
la i udes, and appa en low g ain yield.
Because land aces a e be e adap ed o local en i-
onmen s and a e mo phologically close o desi ed
domes ica ed ypes han wild ba ley, hey a e a mo e
sui able sou ce o gene ic a ia ion o ba ley b eed-
ing. Compa ed wi h he a ia ion in wild ba ley
g own in Finland (AHOKAS and NASKALI 1990),
land aces had signi ican ly highe mean ac i i ies o
a-
and P-amylase and o P-glucanase (AHOKAS and
POUKKULA 1999). The line HA52 was selec ed om
Finnish land aces (AHOKAS 1977), a di e se gene ic
esou ce (AHOKAS and MANNINEN 2001). La e ,
HA52 was ound o ha e a high P-amylase ac i i y
(AHOKAS e al. 1996) and also a high he mos abili y
(AHOKAS and MANNINEN 2000).
The esul p esen ed he e is an ex ension p e ious
s udies o
P-amyl
alleles ound in ba ley (ERKKILA e
al. 1998; ERKKILA 1999). YOSHIGI e al. (1995) de-
sc ibed an allele in c . Ha una Nijo (EMBL-Gen-
Bank da abase accession numbe D49999). We ha e
p e iously ound wo
P-amyl
alleles, one in c .
Ado a and ano he in
H.
spon aneum
s ain PI
296897 (EMBL-GenBank da abase accession num-
be s AF061203 and AF061204, ERKKILA e al. 1998).
All he alleles di e om each o he h ough hei
nucleo ide sequences especially in in on 111, bu no
signi ican ly in he open eading ame (ERKKILA e
al. 1998). Fu he s udies showed ha se e al ba ley
cul i a s, lines and wild s ains had ei he a c .
Ado a-like, c . Ha una Nijo-like o
H.
spon aneum
PI 296897-like
P-amyl
allele (ERKKILA 1999).
To
s udy he
P-amyl
locus in he Finnish land ace ba ley
line ha ing high enzyme ac i i y, he gene o he line
HA52 was sequenced and compa ed wi h p e iously
ound alleles.
The
P-amyl
sequence
in
HA52
-
The locus o
0-amyl
in he Finnish land ace line HA52 was PCR ampli ied
om genomic DNA using p ime s based on he
sequence o he c . Ha una Nijo (YOSHIGI e al.
1995). DNA sequencing was pe o med on ALF
DNA Sequence (Pha macia-LKB) and sequences
we e analysed wi h he PC/Gene so wa e package
(In elliGene ics). The sequence o he HA52
P-amyl
gene ob ained was 4951 bp in leng h and included a
p omo e , se en exons and six in ons, which is con-
sis en wi h he schema ic s uc u e o all he alleles
sequenced be o e (Fig. 1, YOSHIGI e al. 1995;
ERKKILA e al. 1998). The
P-amyl
sequence o HA52
has an EMBL accession numbe AJ301645. T an-
sc ip ion ini ia ion si e is si ua ed a posi ion 1205 bp
om he beginning o he clone.
Some p omo e elemen s, such as TATA and
CCAAT boxes, a e common o many genes an-
sc ibed by polyme ase 11. In he HA52
P-amyl
a
92 M.
J.
E kkila and
H.
Ahokas
He edi as
134 (2001)
Fig.
1.
Schema ic p esen a ion
o
P-amy
I
s uc u al gene.
Se en exons a e ma ked
wi h
black boxes, and
six
in ons
wi h whi e boxes (nume als I-VI). G ay box, ATG and
TAG indica e p omo e , ansla ional s a and s op
codons, espec i ely. The HASZspeci ic dele ions
and
inse -
ions a e ma ked
in
in ons
I1
and
111.
A
segmen
o
p omo e
is
enla ged showing
he
ela i e posi ions
o
ATG
ansla ion s a si e, ansc ip ion ini ia ion si e, TATA
box, I-box, wo GGTTT mo i es and HA52-speci ic
92-bp
dele ion.
TATA box is a
-
31 bp, as can be expec ed, since in
plan s, i is no mally be ween
-
29 bp and
-
33 bp
(MESSING e al. 1983). T ansla ion s a s a posi ion
+
55 bp (Fig. 1) and has he same mo i e as common
consensus sequence
in
plan s, CCACCATG
(KOZACK
1984).
In ons and inse ions
-
Compa ed wi h he alleles o
c . Ha una Nijo, c . Ado a, and
H.
spon aneum
PI
296897 (YOSHIGI e al. 1995; ERKKILA e al. 1998),
he HA52
P-amy
l
allele had se e al subs i u ions, and
addi ionally, bo h single-base and sho mul iple-base
dele ions and inse ions (Table
1).
Majo sequence
di e ences be ween he ou
P-amy
1
alleles we e in
he p omo e egion, and in he in ons I1 and I11
(Fig.
1).
The e we e sho inse ions o
3
bp and o 10
bp close o each o he in he in on I1 o HA52 (Fig.
2B). The 10-bp inse ion con ains a epea ,
5'-
ATATTTA-3', which is u he mo e ound once o
wice in he in ons I11 o he ba leys s udied
so
a .
The in on I1 inse ion in he HA52
P-amyl
is
10
bp
long and con ains he ATATTTA epea (Fig. 2B)
and is also ound wice in i s in on 111, hence sug-
ges ing a con e sion a eplica ion.
GNIADKOWSKI
e
al.
(1
996) sugges ed ha U- ich sequences s imula e
splicing in RNA, ega dless o hei posi ion wi hin
an in on. This holds in dico s, bu he monoco
splicing machine y is less dependen on UA composi-
ion (GOODALL and
FILIPOWICZ
1991). Thus he
sho AT- ich inse ion in HA52, a monoco , in on
I1 has p obably no e ec on ansc ip ion.
Dele ions
-
The e is a dele ion o 21 bp in he in on
I11 o HA52 (Fig. 2C). In c . Ado a, c . Ha una
Nijo and
H.
spon aneum
his agmen con ains a
epea , GGTGGG, which is ound ou imes a he
end o he ORF in all
P-amyl
alleles, also in HA52
allele. The longe agmen in he in on I11 may
se e as a binding si e o a nega i e ansc ip ion
ac o being a eason o he highe p-amylase ac i -
i y in HA52 han he ba leys ha ing he G- ich
epea .
The 92-bp dele ion in he p omo e egion o HA52
(Fig. 2A) is posi ioned
451
bp ups eam om he
TATA
box
(Fig. 1). OKADA e al. (2000) ound wo
di ec epea s in he same egion in c . Ha una Nijo.
The o he segmen s o he epea s a e ela i ely close
o he CCAAT box a
-
194 bp. Only 15 bp up-
s eam om he CCAAT HA52 has an I-box, de ined
as GATAA by TERZAGHI and CASHMORE (1995).
Rela ed mo i es o GATAA a e ound in many plan
p omo e s, some o which a e ligh egula ed ( e-
iewed by
TERZAGHI
and
CASHMORE
1995). The
I-box in
P-amyl
is 183 bp ups eam om he TATA
Table
1.
Numbe
o
he bases in ol ed in he unique al e a ions
o
he P-amyl alleles
in
ba ley
Ba ley To al numbe
o
bases
In dele ions In inse ions
In
subs i u ions
~~
Ado a
0
HA52
125
H.
spon aneum
39
Ha una Nijo
25
Numbe
o
bases
132
28
1
0
8
40
5
7
In he longes dele ion
In
he longes inse ion
Ado a
0
HA52
92
H.
spon aneum
38
Ha una Nijo
25
126
10
0
0
He edi as
134
(2001)
B ie epo
93
A
Ado a
HA5 2
H.
spon aneum
Ha una
Nijo
Ado a
HA5 2
H.
spon aneum
Ha una
Ni
j
o
Ado a
HA5 2
H.
spon aneum
Ha una
Nijo
B
Ado a
HA5 2
H.
spon
aneum
Ha una
Nijo
C
Ado a
HA5 2
H.
spon aneum
Ha una
Nijo
-616
-524
-615
-615
-567
-566
-566
-517
-482
-516
-516
TTTTTTTGGCCCCC-GAAGCATATTCTTCCGGGAGCCAAATTGACATTCC
TTTTTTTGGTCCCTGGAAGCATATTCTCCCTTGAGCCAAATT--------
TTTTTTTGGCCCCC-GAAGCATATTCTTCCGGGAGCCAAATTGACATTCC
TTTTTTTGGCCCCC-GAAGCATATTCTTCCGGGAGCCAAATTGACATTCC
GGTCATGATGTGWTTGGATC-GTTAGTTATACAGATAAGGATATAT
mTACCTCAACCGAATCTAGGTTACAACAAGCTTAACACTCATGCATTAG
..................................
AACATTCATGCATTAG
CPTACCTCAACCGAATCTAGGTTACAACAAGCTTAACACTCATGCATTAG
CPTACCTCAACCGAATCTAGGTTACAACAAGCTTAACACTCATGCATTAG
739
CTAGTTCTCTGATGCATAT-T---TATA----------
TAGAAGTTCAAG
736
CTAGTTCTCTGATGCATATATAGATATACATATTTAGATAGAAGTTCAAG
739
CTAGTTCTCTGATGCATAT-T---TATA----------TAGAAGTTCAAG
739
CTAGTTCTCTGATGCATAT-T---TATA----------
TAGAAGTTCAAG
1982
1871
1835
1877
TGCTTATGGAGAAAGGTQTATGCATTTATACTTCAACAATAAGAATA
TGCTTATGGA---------------------
TACTTCAACAATAAGAATA
TGCTTATGGGGAAAGGTQTATGCATTTATACTTCAACAATAAAAATA
TGCTTATGGGGAAAWTGGGCTATGCATTTATACTTCAACAATAAAAATA
Fig.
2A-C.
Sec ions
o
nucleo ide sequence alignmen s
o
P-a nyl
alleles o c . Ado a, HA52,
H.
spon aneu n
PI
296897
and c . Ha una Nijo.
A
A dele ion
o
92 bp in he p omo e egion o HA52. Palind omic sequences a e in i alics and
in e ed epea s a e in bold ace.
B
Inse ions in in on I1 o HA52 wi h he AT-mo i e in bold ace.
C
A
dele ion
o
21
bp in in on I11 o HA52 wi h he bold GT-mo i e. The sequence in bold ace in
B
also appea s in in on I11 wice, and
he bold sequence in
C
appea s ou imes in owa ds he end o he se en h exon o he
P-amyl
gene.
box (Fig. 1). This is in ag eemen wi h many ligh -
egula ed ibulose- 1,5-bisphospha e ca boxylase
genes ha ing a single I-box 100-300 bp ups eam
om he TATA box (BORELLO e al. 1993).
The dele ed agmen o 92 bp in he p omo e o
HA52 is ele an in he o he 0-amyl alleles. Th ee
in e ed epea s, ou palind omic sequences, and
se en hai pin loops we e ound om he 92 bp
agmen in he p omo e egion in P-amyl o c .
Ado a, c . Ha una Nijo and
H.
spon aneum
PI
296897 (Fig. 2A and da a no shown). This highly
epe i i e sequence in p omo e egion 482 bp up-
s eam o he ansc ip ion ini ia ion si e (Fig.
1)
is a
pu a i e binding si e o a nega i e ansc ip ion ac-
o . I s absence may con ibu e o he high 0-amylase
ac i i y o g ain mass in HA52, being 2.5 imes ha
o c . Ha una Nijo and 2.9 imes ha o c . Ado a
(AHOKAS and MANNINEN 2000).
GGTTT
mo i e and subs i u ions
-
Two GGTTT
mo i es we e ound a posi ion -421 bp and -436
bp om ansc ip ion ini ia ion si e (Fig.
1).
The
GGTTT mo i e and addi ionally a GCCGC mo i e
a e c i ical o exp ession in endospe m and emb yo
in he maize
Adhl
(alcohol dehyd ogenase 1) gene
p omo e exp essed in ansgenic ice (KYOZUKA e
al. 1994). Because hese mo i es a e also equi ed o
exp ession in o he issues, KYOZUKA e al. (1994)
assumed ha he e migh be issue-speci ic pos - ans-
la ional modi ica ions o binding p o eins o possibly
addi ional p omo e elemen s. These mo i es may
also hold ue o he
Adhl
-like genes in o he mono-
co s bu no necessa ily o o he genes. Because no
GCCGC mo i e was ound in
0-amyl
gene, u he
in es iga ions a e needed o explo e i he GGTTT
mo i e (Fig. 1) alone is adequa e o speci ying he
gene exp ession in endospe m. In 0-amyl p omo e ,
94
M.
J.
E kkilu
and
H.
Ahokas
He edi as
134
(2001)
OKADA
e
al.
(2000)
did
no
ind any speci ic
se-
quence simila o he endospe m box, which
is
known
as
a
common sequence
in
he p omo e egion
o
p olamin genes and which
seems
o be associa ed
wi h seed
speci ic
exp ession (HAMMOND-KOSACK e
al. 1993).
Fou single-base subs i u ions we e ound in he
open eading ame o HA52.
All
o hese led o
amino acid subs i u ions. The amino acid subs i u-
ions in
P-amyl
o HA52 a e unique compa ed wi h
known amino acid sequences o c . Ado a, c .
Ha una Nijo, and
H.
spon aneum
PI
296897 namely
A g-115
+
Cys,
Asp-164
--
Glu, Phe-246
+
Leu,
and
Val-430
--
Ala.
None o hese subs i u ions is ound
in conse ed egions
o
in he icini y o ac i e si es.
The amino acids a he posi ions 115,
164
and 430 a e
iden ical
in
c . Ha ing on (c . KANEKO e
al.
2000)
and in HA52. Because he e
is
no P-amylase ac i i y
da a
om
same
ial, we can only specula e
on
he
ole o he wo amino acid di e ences, Phe-246
+
Leu
and Th -520
--
Ala, be ween c . Ha ing on and
HA52. The p omo e egion and he in ons p obably
ha e
mo e
e ec on he P-amylase ac i i y han
amino acid subs i u ions, al hough hese may a ec
seconda y modi ica ions o a ini ies
o
he p o ein.
The Canadian c . Ha ing on has ances o s among
Swedish
and
No wegian land aces. The e is
a
25%
chance ha c . Ha ing on inhe i ed i s
P-amy
1
allele
om
c .
Bjmneby, p o ided ha he e was
no
in a-
genic ecombina ion. C . Bj~ neby o igina ed om
he a ea o T ysil, No way (HAUGUM 1940). A ound
1600 Finns, especially om Sa onia, a sou h-eas e n
p o ince
o
Finland,
immig a ed o his
a ea
whe e
hey used slash-and-bu n cul i a ion
(HANSEN
1904;
HAMALAINEN
1947). The Finns b ough ce eal
seeds
wi h hem (NORDMANN 1888;
HAMALAINEN
1947).
The e o e, he c . Bja neby and he line
HA52
may
ep esen
a
common gene
pool
om
SE Finland.
O he simila i ies
o
hei
P-amyl
genes would be
wo h compa ing.
The p omo e egion lacking om
he
HA52
P-
amyl
allele p o ides g ounds o pos ula e ha his
egion se es
as
binding si e o
a
nega i e egula ion
ac o . The HA52-speci ic p omo e and amino acid
sequence
may
also
cumula e in he P-amylase ac i i y.
Compa ison be ween he di e en
P-amy
1
alleles and
P-amylase
ac i i ies sugges ha he p omo e and he
in on egions exe
mo e
in luence on
P-amyl
gene
exp ession han he obse ed amino acid changes.
Fu he s udies o
he
p omo e egion a e needed o
con i m p esen pos ula ions and o de e mine spe-
ci ic egions
o
enhancing, ep essing and
endospe m-
speci ic exp ession.
ACKNOWLEDGEMENTS
We hank Ms A Vi a o he ALF ope a ions. This
esea ch was unded by he Raisio Yh yman Tu kimussaa-
i6 Founda ion and Finnish Cul u al Founda ion.
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