ArticlePDF Available

Variations in Endogenous Gibberellins in Developing Bean Seeds I. Occurrence of Neutral and Acidic Substances

Authors:

Abstract and Figures

Activities of separated and chromatographed substances in the nonacidic, acidic ethyl acetate and acidic butanol fractions from bean seeds, Phaseolus vulgaris L., cv. Bountiful and Kentucky Wonder, were measured in the Progress No. 9 dwarf pea bioassay grown under red light. Activity in the nonacidic fraction was shown to be attributable only to neutral substances and was free of acidic gibberellin-like substances. As the seed matures, neutral substances and one of the acidic butanol-soluble substances (B-I) increase in activity. The acidic ethyl acetate substances and butanol-soluble substance (B-II) initially increase and then almost disappear.
Content may be subject to copyright.
Plant
Physiol.
(1966)
41,
623-628
Variations
in
Endogenous
Gibberellins
in
Developing
Bean
Seeds
I.
Occurrence
of
Neutral
and
Acidic
Substances'
Tohru
Hashimoto2
and
Lawrence
Rappaport
Department
of
Vegetable
Crops,
University
of
California,
Davis
Received
September
27,
1965.
Summary.
Activities
of
separated
and
chromatographed
substances
in
the
nonacidic,
acidic
ethyl
acetate
and
acidic
butanol
fractions
firom
bean
seeds,
Phaseolus
vulgaris
L.,
cv.
Bountiful
and
Kentucky
Wonder,
were
measured
in
the
Progress
No.
9
dwarf
pea
bioassay
grown
under
red
light.
Activity
in
the
nonacidic
fraction
was
shown
to
be
attributable
only
to
neutral
substances
and
was
free
of
acidic
gibberellin-like
substances.
As
the
seed
imatures,
neutral
substances
and
one
of
'the
acidic
butanol-soluble
substances
(B-I)
increase
in
activity.
The
acidic
ethyl
acetate
substances
and
butanol-soltuble
sub-
stance
(B-II)
initially
increase
and
then
almost
disappear.
Changes
in
levels
of
gibberellin-like
substances
during
ontogeny
of
seeds
and
buds
are
well
docu-
mented.
These
substances
are
present
in
comparative
abundance
in
immature
seeds,
but
decrease
in
amount
as
the
seeds
mature
(1,
7,
8,
11,
12,
17,
18).
Acidic
gibberellins
are
present
in
high
concenttration
in
po-
tato
stolons,
but
essentially
disappear
when
the
tubers
form
(19).
They
are
present
only
in
small
qu'antity
in
buds
of
resting
potato
tubers,
but
they
increase
in
level
as
the
tubers
sprout
(13,
15,
19,
20).
Ree-
ogni'tion
of
these
changes
led
us
to
the
hypothesis
(5)
that
gibberellins
present
in
plant
organs
during
periods
of
active
growth
are
converted
into
forms
that
are
relatively
inactive
during
periods
of
arrested
growth.
Radley
(14),
Corcoran
and
Phinney
(1),
and
Skene
and
Carr
(18)
have
shown
that
gibberellins
extracted
by
the
usual
method,
i.e.,
At
low
pH
(2.5-
3.0)
in
ethyl
acetate,
increase
to
a
peak
in
the
imma-
ture
bean
seed
and
then
disappear
as
the
seed
dries.
However,
Skene
(16)
and
Hashimoto
and
Rappaport
(2)
found
both
acidic
and
nonacidic
giibberellin-like
substances
in
green
'bean
seeds.
The
latter
investi-
gators
also
showed
that
nonacidic
gibberellin-like
substances
occur
even
in
the
mature
dry
seed;
these
com,pounds
may
resemble
neutral
substances
reporte(d
to
occur
in
peelings
of
potato
tubers
(3,4).
Addi-
tionally,
Hashimnoto
and
Rappaport
found
that
gib-
berellhin-ike
substances
Which
are
insoluble
in
ethyl
acetaite
but
soluble
in
butanol
occutr
in
bean
seedls
at
various
stages
of
development.
This
paper
presents
a
new
analysis
of
clhanges
in
gibberellin-l'ike
substan,ces
during
ontogeny
of
the
l)ean
seed.
Materials
and
Methods
Seed
Materials.
Seeds
of
Phaseolus
vulgaris
L.,
cv.
Kentucky
Wonder
and
cv.
Bountiful
were
used.
All
seeds
were
harvested
from
plants
grown
at
the
University
Farm
at
D'avis,
except
the
mature
dry
seeds
of
Bountiful.
These
were
obtained
from
com-
mercial
sources.
Bean
fruits
were
harvested
and
selected
for
uni-
formity;
the
seeds
were
removed
from
the
pericarp
as
quickly
as
possible.
Seeds
of
Bountiful
were
divided
according
to
length
land
weight
into
3
arbi-
trary
developmental
stages:
immature,
mature
green,
and
mature
dry.
Developmental
stages
assigned
to
Kentucky
Wonder
were
medium
mature
and
mature
green.
The
length
and
fresh
weights
of
the
seeds
are
'shown
in
table
I.
Seed
size
is
not
a
precise
estimate
of
age
but,
nevertheless,
is
useful
as
all
index
to
the
stage
of
development
and
as
a
basis
for
comparing
data
with
that
of
other
workers.
Extraction
and
Fractionation.
These
procedures
were
similar
to
those
used
by
Haya'shi
and
Rappa-
port
(3,
5).
The
seeds
were
covered
with
methanol,
ground
in
a
Waring
blendor
for
5
minutes,
and
the
homiiogenate
filtered
through
a
Buchner
funnel.
The
extraction
was
repeated
by
washing
the
residue
with
T
able
I.
Length
and
Fresh"
Weight
of
the
Seeds
Data
concerning
Kentucky
Wonder
and
Bountiful
bean
seeds
of
the
stages
of
development
studied:
immature
(I),
mediumi
mature
(MM),
mature
green
(MG),
and
nmature
dry
(MD).
The
fresh
weight
of
MD
seed
was
measured
after
imbibition
in
water.
Kentucky
wonder
MM
MG
Bountiful
I
MG
MD
1
This
investigation
was
supported
in
part
by
Grant
No.
EF-61
of
the
United
States
Public
Health
Service.
2
Present
address:
Department
of
Agricultural
Chem-
istry,
University
of
Tokyo,
Bunkyo-ku,
Tokyo,
Japan.
Length
(mm)
Fr
wt
(mg/seed)
Dry
wt
(mg/seed)
623
11
142
28
17
517
134
9
73
9
16
635
201
19
1100
443
PL4
PLANT
PHYSIOLOGY
methanol.
Mat
atuire
(lr\
seed
\\
as
extracted
xvith
50
%
ihethanool.
The
extract
\\,as
e\aploratecd
to
the
water
phase
tln(ler
reduced
pressuee.
Tl'
o
mkake
partitioning
as
(Itlatitative
as
)ossil)ble,
the
water
phase
wvas
supple-
mlente(1
w
vith
\\vater
xxe
llh
len(need,
prociding
a
ratio
of
about
150
nl
to
50g
o
f
seeds
fresh
weight.
The
\\water
phase
xvas
a(lj
uste(d
to
p14
7.5
\\ith
NaHCO.
ail(l
extracted
3
tim,es
xxith
equial
vomnmes
of
ethyl
acetate
wvhich
\\,ere
conll)ined.
This
\-as
the
non-
acidic
fractioni.
The
remiainiinig
wvater
phase
was
adjtiste(l
to
pH
3.0
xxith
(lilute
H,PO,
and(I
shaken
3
timies
xvith
ethyl
acetate.
T'hiis
gave
the
acidic
ethyl
acetate
fraction.
Since
gibberellin
activity
wvas
founid
in
thle
remaining
xxwater
phase,
it
was
further
extracte(l
wvith
n-hutanol.
giving
the
acidic-butanol
fractioni.
'I'h
e
3
fractionis
thus
ob)taine(l
were
de-
hydrated
by
passing
through
aun
hydlrouis
Na.SO4
colunmns
anldi
xere
conicentrated
under
reduced
pres-
sure.
.\dditional
details
of
fractioniationi
are
supplied
i
the
(lisclussion
of
ii(liiual
exlperiments
tunder
Rtesul
s.
Papcr
('Co;oiatoyraph
'Iv.
'he
fractions
were
strea.ked
oni
\Vhatn,an
N\o.
3
MM\
paper
anid
chro-
matogra)he(l.
Ai
isopropyl
alcoholannuoniuni
hy-
(Iroxi(le
(28
%
)
-wvater
(10:1
v
i
)
s
tiolui
wvas
the
develo)ping
solvent
for
the
aci(lic
ethvl
acetate
and
lbutanl
fractions.
A
soltitiionl
in
which
n-butyl
alcohol
\x\as
substituted
for
the
isopropvl
alcohol
was
tused
\\ith
the
nionaci(lic
fr-actioni.
The
solvent
front
xas
allo\ved
to
runlc
a
(listance
of
23
cmil
in
(lescending
chromiatograplhy
at
25;.
'T'he
chromiatogram
then
xvas
(lrie(l
and
cuit
inito
10
equal
sectionis
l)arallel
to
the
origini
at
each
).1
RI,
value.
Sometimes
the
origin
wXas
used
as
ll
aadditional
sectioni.
The
ma-
terial
oni
each
section
(2.3
>\
57.3
cmi
)
wx'as
eluted
with
absolute
methaniol
i
(lescenidinig
ipaper
chrotma-
tograplhy
for
2
or
3
(lays,
and(
the
eltuates
that
drip)ped
off
the
enid
of
the
stripxs
xvere
collecte(l.
The
final
volum,,e
of
each
eluate
\x\as
about
15;
nil.
'T'he
eluates
xere
(Iried
unider
reduce(d
pressure
ail(l
prep)ared
for
bioassay
by
dlissolving
ill
a
solutioin
of
eqjual
volumes
of
ethanol
anid
vater
containin'g
0.5;
%
Tween-20
(
polvoxvethylene
sorbitan
imolnolatrate
)
\hen
ac-
tivitv
ill
1
RF
zonie
is
(liscussed.
ollyx
the
upper
edge
of
the
his.togrami
is
rel)orte(l.
If
activity
occnrre(l
ox-er
miore
than
1
14,.
zonle,
the
lo\\xer
ati(l
ulpper
edlges
of
the
enitire
zonie
are
nm,entioned.
Bioa
sav.
The
l)e
blioalssaV
usel
in
thlis
.stud-
\x\as
originatedl
h)v
E.
Rteinhard
andcl
A.
Lang
(unpub-
lished
)
and
was
iutilize(d
1by
Ken(le
and
Lanig
(7).
It
\\
aIs
iio(lified
bx
Ibv
a,ashi,
l3lti,enthd
I-(;ol(lschmiiidt.
an(l
zal)p)aport
(4).
Seed.s
of
(dwarf
pleas
(Pisum111
sativnuin
14.,
cv.
Progress
'No.
9)
were
soaked
for
6
to
8
hours
in
runuing
tal)
water,
sowvn
ill
trays
of
\\et
vermiculite,
and
allowe(d
to
germvinlate
ill
total
darkness
for
4
(lays
at
23
to
25°.
Theni
seedlings
measuirilng
2.75
to
3.25
cm
from
the
cotvledolnarv
node
to
the
hook
xvere
transferred
to
trays
wvith
water
at
23
to
25°
uinder
red
light.
The
recl
light
xas
obtained
1v
filteriiig
the
light
frollm
fotlr
40
-x-
fluiorescenit
tuibes
throug-h
3layers
of
red
cellophane.
Ilased
oni
spec-
trophpotometiric
examlination
(If
the
cellophanhe,
the
bulk
of
traiisnsittedI
light
xvas
In
the
640-
to
700-nii,,
regiion.
.
'TIvet
ltyfour
hrouirs
after
transfer
(If
seedl
hlusgs,
5/J
(f
test
soluition
vere
appIliedl
xxvith
a,
miicro
sy
ring-e
to
tIe
apex
of
each
plant.
Thehe
leigtll
of
tIe
shoot
froiul
the
cotyledolinarv
ilo(le
to
the
hi.hest
-is
ible
ilo(le
xvas
measuredS
day
s.
after
treatllment.
D)ata
for
elongation
t
xxere
explresse(l
accordll-g
to
the
following
equation
1
1ercenit
of
control
Length
conttrol
pllaint
-3(0
cm
Length
treated
pllanlt
3.0
ciii
1
I00.
TIhe
3.0
cmi
values
represent
the
taxerage
jiiitial
length
of
the
seedling
s.
A
standard
curve
of
activity
ff
o
(
A
is
shoxvn
i
figure
1.
I)osage
is
exlpressedl
as
extract
equivalent
to
x
number
of
seeds
or
extract
equivalent
to
x
g
of
seeds.
In
fig,ures
2
andl
3
the
5
%
coilfidenice
Iiimts,
deter-
mileled
taccor(linio
I
Sile(lecor
(21
).,
are
in(dicatedI
l)v
uniisha(led
areas.
'I'he
lhade(ld
areas
showx
statisticalkx
sigificant
acti\tit-.
DWARF
PEA
BIOASSAY
*
123/62
12/162
662
-
60
0
-
12/
23/
62
-
o
21218/63
C-
500i
*
2
*
2/2
5/
6
4o
o
3
/6
31
3
?
3
19
63
3r26
3
o
31
D
z
<
/
°
Ci)
20
/
00025
0
0
25
LU
G
GA
/PLANT
Fi(i.
1.
Response
cuirve
of
Progress
No.
9
(lx\,arf
peas
groxxl
unde(ler
re(l
light
folliving
treatIlleilt
\x
ith
several
coniceIltrationis
o)f
G(A.
Poiilts
relpreselnt
valutes
of
observations
ma(le
in
8
tests
over
a
perio(l
of
4
illoiiths.
All
c
cenltratiolns
\\xerc
ilot
teste(
in
eaclh
test.
Results
,(.j6j.
i+'tiIl
.
cc/ole
,Pracetioo.
'l'lhe
lative
-ib-
lherellins
il
lliattire
(Irv
see(d
\x
er-c
ex.allined
first.
1101'
bioassav,
aii
eluiate
from3
ain
R1
zolie
of
tlhe
chroma-
to(,raill
ol)taine(l
from
z
ani
extract
e(quivalent
to
5
g
of
see(l
xxas
applied
to
each
pea6
lalalnt
(fig
2.
uIlpper
hi.to(railams
)
lll
eorigiial.
extract
as
(liluite(I
3)
tines.
al(ld
the
(lata
are
shownl
il
the
lowver
Ilisto-
grail.s.
Ill
the
origilnal
acidic
ethlvI
acetate
fractioni,
activitv
was
found(I
at
RF
0.1.
0.3,
and
0.5.
I
n
the
lox-er
Ilistogralii
slight
activity
is
apparent
at
R,(
0.2.
'rlhe
total
activity
in
the
aci(dic
eth\I
1
aicetate
fi-actioi
624
HASHIIMOTO
AND
RAPPAPORT-GIBBERELLINS
IN
B3EAN
SEEDS
220"
NON-ACIDIC
200F
Ethyl
Acetate
sBob
166F
140F
120[
2
r
Ii
TT.
iT
ACIDIC
Ethyl
Acetate
Original
Extract
_.I!
K
H
[4
C
-.
T
T
TTT
T
IMMATURE
:
ACIDIC
-Butanol
;H
m
,--t.
CDA
x
4
MH
7
.i,
C
iI
TT
T
T
-0
2
4
6
8
10
0
2
4
8-
I.0
-0
I
I.-
0
.2
4 6
8
1.0
0
.2
4
.6
8
1.0
0
.2
4
.6
8
130
180I
Diluted
3x
160r
140-
120~
K-
oo
L
L
80i
..
.
.
.
0
.2
4
6
8
1
F
.i
1
*
11
L
-
ci
0
2
4
6
8
10
0
2
4
6
8
10
Rf
FIG.
2.
Response
of
Progress
No.
9
pea
planits,
grown
under
red
light,
to
gibberellin-like
substances
ex-
tracted
from
mature
dry
seeds
of
Phascoluts
,idlgaris
L.,
cv.
Bountiful.
Dosage:
Original
extract
represents
5
g
of
seed
equivalent
fresh
weight;
10
plants
per
treat-
ment.
Shaded
portions
show
activity
above
the
5
%
confidence
limits
of
the
control.
The
narrow
strip
to
the
left
of
the
acidic
ethyl
acetate
and
butanol
fractions
represents
activity
of
the
origin
of
the
chromatogram.
T
refers
to
toxicity
symptoms.
I
1-J
z
CD
C-)
C)
z
1
C)
-j
LUJ
80
MATURE
GREEN
r_
4:x
CD
LC)
C)
c;
.4
0.
.
.
.
0
.2
4
.6
.8
1.0
0
.2
4
.6
.8
1JD
Rf
FIG.
3.
Activity
of
gibberellin-like
substanices
in
the
acidic
butanol
fraction
of
immature
(9
mm)
and
mature
green
(16
mm)
seeds
of
cv.
Bountiful
as
measured
on
Progress
No.
9
pea
plants
grown
under
red
light.
Each
of
10
plants
received
extract
equivalent
to
0.5
g
of
seed.
Shaded
portions
show
activity
above
the
confidence
limits
of
the
control.
GA1
markers
show
the
position
to
which
authentic
GA1
traveled
in
co-chromatography.
T
refers
to
toxicity.
weight
(lecreased
with
maturation,
but
activity
per
seed
increased
as
the
seed
developed.
Activity
of
B-II
decreased
on
a
fresh
weight
basis,
and
activity
per
seed
was
low
in
immature
seed,
high
in
mature
green
seed,
and
decreased
as
the
seed
matuired1.
was
very
low,
corresponding
to
the
(lata
reporte(l
several
workers
(1,8,
11,
12,
16,
17,
18).
Acidic
Butanol
Fraction.
The
exceedingly
highl
activity
in
the
butanol
fraction
(fig
2)
is
miiost
strik-
ing,
especially
since
the
fraction
was
extracte(d
from
(lry
seed.
The
activity
at
zone
RF
0.0
to
0.4
was
especially
strong,
even
after
diltition.
Since
toxicity
(T)
appeared
in
somile
zones
of
the
upper
hi.stogranm.
the
lower
histogram
is
considere(d
to
lpresent
a
miore
reliable
picture
of
the
distribution
of
acti\ifty
althouigli
(lilution
reveale(d
n1o
important
qtualitative
(lifferel
ces.
Zones
of
activity
were
present
'at
RFS
0.2
and
0.5
in
immature
seed
and
at
RFS
0.2
anid
0.3
to
0.5
in
mlature
green
seed
(fig
3).
These
zones
prol)ably
contain
mnore
than
1
active
substance.
However,
for
convenience
the
lower
and
upper
RF
zones
in
both
stages
of
development
are
designlated
as
substance
B-I
and
substance
B-Il,
respectively.
Quantitative
changes
in
B-I
and
B-Il
eluates
from
immature,
mature
green
and
mature
dry
seed
of
cv.
Bountiful
were
tested
on
thepea
bioassay
(fig
4).
This
figure
shows
that
activity
of
B-I
per
g
fresh
.02-
LJ
LL
.01
0
B-l
B-Il
_
IMMATURE
MATURE
MATURE
IMMATURE
MATURE
MATURE
GREEN
DRY
GREEN
DRY
FIG.
4.
Activities
of
butanol-soluble
gibberellin-like
substances
B-I
and
B-II
at
3
stages
of
seed
development
of
cv.
Bountiful.
Data
are
expressed
on
per
seed
and
on
fresh
weight
bases.
Bioassay:
Progress
No.
9
dwarf
peas
grown
under
red
light,
10
plants
per
treatment.
-j
C-)
2
G
LL.
C.,
w-
L
625
A919
jig/Seed
PLANT
PHYSIOLOGY
,/7mm
NEUTRAL
...
~~~~~0
,,P
H
mm
NEU
TRAL
/7mm
BASIC
*
//
mm
BASIC
_.
--_
A
_
_a
_
_:_
A--d
0.1
10
SEED
NUMBER
EQUIVALENT/
PEA
PLANT
Table
I
7Jest
for
Occiurr-enict
of
Acidic
Substmiccs
in
tf/2iC
Omiicidic
Fra4
cti(onl
ihle
water
phase
of
the
methanol
extract
of
bean
seeds
was
reextracted
with
ethyl
acetate
at
pH
8.0,
yielding
the
nonacidic
fraction.
Active
chromatographic
eluates
were
reextracte(l
by
the
samie
procedure
anid
a
dilutioni
series
wxas
run.
The
original
eluate
anid
its
(lilutionis
were
testedl
on
Progress
No.
9
dxarf
peas
growni
uinder
re(l
liglht,
10
p)lants
per
treatment.
Noonacidic
eluates
ani(l
cultivar
R
F
0.2-0.4
Kenttucky
wonder
RF.
0.0
Bountiful
RF
0.8
Bounitifil
Dilution
pH
1
1/2
113
8.0
3.01
8.1)
3.0
8.(
3.0
136
113
158
100
128
102
(%
of
conitrol)
100
106
..
..
128
99
144
111
FIG.
5.
Response
of
Progress
No.
9
dwarf
peas
to
unchromatographed
extracts
of
the
nieutral
and
basic
fractions
fromn
mediunm
mature
and
mature
green
seed
of
Kentucky
Wonder.
Bioassay:
Progress
_No.
9
dwarf
peas
grown
under
red
liglht,
10
plants
per
treattmienit.
Nonacidic
Fractiom.
Distinictly
active
zonies
are
seen
in
the
nonacidic
othyl
acetate
fraction
fromii
dry
seed
(fig
2).
In
the
tpper
histogrram
they
occuir
at
RF
0.5
to
1.0.,
and
in
the
lower
histogramii
at
RFS
0.5
(N-I)
an(d
0.7
to
0.9
(N-III).
'T'he
small
(liscrep-
aincies
in
RF
values
in
the
original
and
dilute(d
extracts
probably
are
attributable
to
toxicity.
\Vhen
levels
of
the
nonacidic
gibberellin-like
suib-
stanlces
in
nonlchroniiatographed
extracts
of
meditlumi
mature
ani(
mlatture
greeni
seeds
were
coml)ared
(fig
5),
it
was
apparent
that
the
large
see(ds
colntained
abotut
10
times
more
activity
thall
did
the
me(liu
matture
seeds.
Does
the
Nonacidic
Fractiown
Coto
ain
Acidic
Suib-
stances?
1)espite
intensive
efforts
to
separate
neutral