APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
No .
1991,
p.
3378-3382
Vol.
57,
No.
11
0099-2240/91/113378-05$02.00/0
Copy igh
C
1991,
Ame ican
Socie y
o
Mic obiology
Gibbe ellins
and
Ca o enoids
in
he
Wild
Type
and
Mu an s
o
Gibbe ella
ujiku oi
REYES
CANDAU,
JAVIER
AVALOS,
AND
ENRIQUE
CERDA
OLMEDO*
Depa amen o
de
Gene ica
y
Bio ecnia,
Uni e sidad
de
Se illa,
Se ille,
Spain
Recei ed
19
Feb ua y
1991/Accep ed
16
Augus
1991
A
new
sc eening
p ocedu e
was
used
o
isola e
14
gib
mu an s
o
Gibbe ella
ujiku oi
wi h
modi ica ions
in
he
p oduc ion
o
gibbe ellins.
The
p oduc ion
o
ca o enoids
and
gibbe ellins
was
in es iga ed
in
he
gib
mu an s
and
in
ep esen a i e
ca
mu an s
wi h
a ious
modi ica ions
o
ca o enoid
biosyn hesis.
The
de e mina ions
o
gibbe ellins
we e
ca ied
ou
wi h
a
simpli ied
luo escence
me hod.
One
o
he
mu an s
lacked
bo h
gibbe ellins
and
ca o enoids.
In
many
mu an s
he
wo
pa hways
compensa ed
each
o he :
an
inc ease
in
he
p oduc ion
o
one
g oup
o
compounds
was
accompanied
by
a
dec ease
in
he
p oduc ion
o
he
o he .
Unde
ce ain
condi ions
he
compensa ion
was
quan i a i e
when
he
ou pu
o
he
wo
pa hways
was
measu ed
in
moles
o
he
common
p ecu so ,
ge anylge anyl
py ophospha e.
a-Picoline,
an
inhibi o
o
lycopene
cyclase
in
G.
ujiku oi,
inhibi s
gibbe ellin
biosyn hesis.
O he
agen s
ha
a ec
he
accumula ion
o
ca o enoids
ha e
no
no iceable
e ec
on
he
accumula ion
o
gibbe ellins;
such
is
he
case
wi h
diphenylamine
and
,-ionone,
wo
inhibi o s
o
phy oene
dehyd ogena ion,
and
isible
ligh ,
which
s imula es
ca o enogenesis.
The
gibbe ellins
a e
a
g oup
o
na u al
plan
ho mones
wi h
a ious
e ec s
on
g ow h
and
mo phogenesis
(13)
and
p ac ical
applica ions
in
ag icul u e
and
b ewing.
The
gib-
be ellins
a e
indus ially
pu i ied
om
he
cul u e
media
o
some
s ains
o
he
ascomyce e
Gibbe ella ujiku oi
(6,
12),
a
ice
pa hogen
whose
impe ec
s age
is
known
as
Fusa ium
monili o me.
Te penoids
a e
a
la ge
amily
o
na u al
compounds
ha
includes
he
gibbe ellins,
he
ca o enoids,
which
a e
wide-
sp ead
pigmen s
made
by
plan s
and
by
many
ungi
and
bac e ia,
and
he
s e ols,
which
a e
uni e sal
componen s
o
cell
memb anes.
Gibbe ellins
and
ca o enoids
a e
made
om
a
common
20-ca bon
p ecu so ,
ge anylge anyl
py ophos-
pha e
(11).
The
mycelia
o
G.
ujiku oi
con ain
he
o ange
ca o enoid
neu ospo axan hin
and
i s
p ecu so s,
such
as
o ulene,
-y-ca o ene,
and
phy oene.
The
ca o enoid
concen-
a ion
depends
on
he
cul u e
condi ions,
pa icula ly
he
medium,
illumina ion,
and
empe a u e
(3,
4).
The
p oduc-
ion
o
ca o enoids
is
inhibi ed
by
he
addi ion
o
dipheny-
lamine,
a-picoline,
o
,B-ionone
(2).
Many
colo
mu an s
o
G.
ujiku oi
show
al e a ions
o
ca o enoid
biosyn hesis
(1).
The
deep-o ange
mu an s
ha e
much
mo e
neu ospo axan hin
han
does
he
wild
ype.
Albino
mu an s
lack
ca o enoids
o
accumula e
he
colo less
phy oene.
Red
mu an s
accumula e
o ulene.
Pale-o ange
mu an s
a e
simila
o
he
wild
ype
in
he
ligh
and
accumu-
la e
app eciable
amoun s
o
ca o enoids
in
he
da k
(3).
Indus ial
esea che s
ha e
es ablished
he
condi ions
o
gibbe ellin
p oduc ion
and
ha e
looked
o
high-yielding
s ains.
Ve y
li le
is
known
abou
he
gene ics
o
G.
u-
jiku oi,
and
only
a
ew
mu an s
wi h
speci ic
al e a ions
in
gibbe ellin
biosyn hesis
ha e been
desc ibed
(5).
We
de eloped
simpli ied
me hods
ha
allowed
us
o
isola e
mu an s
de ec i e
in
gibbe ellin
p oduc ion.
Analyses
o
hese
mu an s,
he
wild
ype,
and
mu an s
wi h
al e ed
ca o enoid
p oduc ion
illus a e
he
ela ionships
be ween
he
gibbe ellin
and
ca o enoid
pa hways.
*
Co esponding
au ho .
MATERIALS
AND
METHODS
S ains
and
gene al
p ocedu es.
The
s ains
used
in
his
wo k
a e
lis ed
in
Tables
1
and
2.
Unless
o he wise
s a ed,
cul u es
we e
g own
a
30°C
in
an
o bi al
shake
(120
pm)
in
he
da k
in
500-ml
E lenmeye
lasks
wi h
250
ml
o
liquid
10%
ICI
medium
(10),
a
medium
o
gibbe ellin
p oduc ion
wi h
0.48
g
o
NH4NO3
pe
li e .
Whe e
indica ed,
he
glass
op
o
he
incuba o
allowed
exposu e
o
he
cul u e
lasks
o
whi e
ligh
( luence
a e,
2
W
m-2)
om
i e
luo escen
lamps
(Syl ania
F40T121D).
o-Picoline
was
dissol ed
in
dis illed
wa e
and
added
o
he
medium
a
a
inal
concen-
a ion
o
2
g
li e -1
ei he
immedia ely
be o e
inocula ion
o
a e
9
days
o
g ow h.
Diphenylamine
was
dissol ed
in
96%
e hanol
and
added
o
9-day-old
cul u es
a
inal
concen-
a ions
o
20.3
mg
o
diphenylamine
and
0.6
ml
o
e hanol
pe
li e .
3-Ionone
was
emulsi ied
in
a
mix u e
o
e hanol
and
polyoxye hyleneso bi an
monoolea e
(Tween-80)
and
added
o
9-day-old
cul u es
a
inal
concen a ions
o
0.2
g
o
P-ionone,
2
ml
o
e hanol,
and
1
ml
o
Tween-80
pe
li e .
The
glucose
concen a ions
in
he
medium
we e
de e -
mined
wi h
a
glucose
analyze
(model
27;
Yellow
Sp ings
Ins umen s,
Yellow
Sp ings,
Ohio)
o
wi h
glucose
oxidase
(9).
Ca o enoid
analyses
we e
ca ied
ou
as
desc ibed
p e i-
ously
(2).
The
e m
"in e media e
ca o enes"
designa es
he
sum
o
colo ed
ca o enes;
i
does
no
include
neu ospo a-
xan hin.
Fo
he
compa ison
o
ca o enoid
and
gibbe ellin
p oduc ion,
i
was
assumed
ha
1
mol
o
he
p ecu so
ge anylge aniol
yields
267
g
o
phy oene,
265
g
o
in e me-
dia e
ca o enes,
o
250
g
o
neu ospo axan hin
(hal
o
a
mole
in
each
case)
o
345
g
o
gibbe ellins
(a
mole
o
gibbe ellic
acid).
Fluo ime ic
de e mina ion
o
gibbe ellins.
Fluo escen
de-
i a i es
o
gibbe ellins
we e
ob ained
by
shaking
oge he
0.2
ml
o
cul u e
medium,
0.2
ml
o
e hanol
(96%,
ol/ ol),
and
2
ml
o
a
cooled
mix u e
o
equal
olumes
o
sul u ic
acid
and
96%
e hanol
and
incuba ing
he
eac ion
mix u e
a
48°C
o
30
min.
Fluo escence
spec a
we e
ob ained
wi h
an
LS-5
Pe kin-Elme
luo ime e .
The
luo escence
emissions
a
464
nm
o
samples
exci ed
a
406
nm
we e
compa ed
wi h
ha
o
au hen ic
gibbe ellic
acid
(Sigma
Chemical
Co.,
S .
3378
GIBBERELLINS
AND
CAROTENOIDS
IN
GIBBERELLA
MUTANTS
3379
TABLE
1.
P oduc ion
o
gibbe ellins
and
ca o enoids
in
he
wild- ype
IM158289
and
in
gib
mu an sa
Gibbe ellins
(mg
Ca o enoids
(jig
g
o
d y
w -')
S ain
6
days
12
days
Phy oene
In e media e
Neu ospo-
ca o enes
axan hin
IM158289
22 79
1
6
3
SG121
3
10
0
11
7
SG122
7
27
2
19
12
SG123
1
15
09
3
SG124
2
25
0
5
3
SG127
5
15
0
0
0
SG128
6
32
0
8
4
SG129
7
23
0
3
2
SG133
15
49
0
2
1
SG134
10
57
0
6
1
SG135
4
5
1
18
6
SG136
4
82
22
15
SG137
00
0
6
1
SG138
00
2
19
16
SG139
0
1
2
14
9
a
All
cul u es
we e
incuba ed
a
30'C
in
he
da k.
Cul u es
used
o
gibbe ellin
analyses
we e
g own
in liquid
medium.
Those
used
o
ca o enoid
analyses
we e
g own
on
minimal
aga
o
4
days.
Louis,
Mo.)
ha
had
been
dissol ed
in
0.2
ml
o
e hanol
and
mixed
wi h
esh
medium
and
e hanolic
sul u ic
acid
as
desc ibed
abo e.
The
de e mina ions
o
s anda d
and
p ob-
lem
samples
we e
un
in
pa allel.
The
alues
gi en
a e
he
a e ages
o
a
leas
wo
de e mina ions.
Rapid
sc eening
o
gibbe eilin
p oduc ion.
The
colonies
o
be
sc eened
we e
ans e ed
indi idually
o
ials
wi h
1
ml
o
liquid
medium
and
incuba ed
o
9
days
a
30°C.
A
0.5-ml
sample
o
cul u e
medium
was
slowly
added
o
a
ube
wi h
0.25
ml
o
sul u ic
acid
and
obse ed
unde
a
"black-ligh "
lamp
(Syl ania
F18W/BLB/ES).
The
p esence
o
gibbe ellic
acid
in
he
sample
led
o
a
blue-g eenish
luo escence.
Mu agenesis.
A
wa e
suspension
o
IM158289
spo es
was
exposed
o
1
h
a
22°C
o
0.2
mg
o
N-me hyl-N'-ni o-N-
ni osoguanidine
pe
ml
(1),
washed,
and
pla ed
on
minimal
aga
wi h
1
g
o
yeas
ex ac
pe
li e .
The
esul ing
colonies
we e
inocula ed
wi h
oo hpicks
on o
minimal
aga ,
incu-
ba ed
o
3
days
a
30°C,
and
hen
ans e ed
o
liquid
medium
o
he
apid
sc eening
p ocedu e.
The
mycelia
whose
cul u e
media
showed
educed
o
al e ed
luo escence
we e
ans e ed
o
esh
minimal
aga
and
allowed
o
spo ula e
o
a
epe i ion
o
he
sc eening
p ocedu e.
RESULTS
Simpli ied
me hod
o
gibbe ellin
analysis.
The
a ailable
me hods
o
he
analysis
o
gibbe ellin
con en
a e
oo
cumbe some
o
he
quick
sc eening
o
many
samples.
We
de eloped
a
simpli ied
me hod
based
on
he
luo escence
o
he
eac ion
p oduc s
o
gibbe ellins
and
sul u ic
acid.
An
aliquo
o
he
Gibbe ella
cul u e
medium
is
incuba ed
wi h
an
e hanol
solu ion
o
sul u ic
acid.
The
ligh
emission
a
464
nm
is
de e mined
in
a
spec o luo ime e
a e
exci a ion
a
406
nm
and
compa ed
wi h
ha
o
a
pu e
gibbe ellic
acid
s anda d
(Fig.
1).
The
emission
and
exci a ion
luo escence
spec a
ob-
se ed
a e
applica ion
o
his
me hod
o
he
cul u e
media
o
he
wild- ype
IM158289
and
he
deep-o ange
mu an
SG22
coincided
wi h
ha
o
pu e
gibbe ellic
acid.
The
esul s
o
his
me hod
we e
in
good
ag eemen
wi h
hose
ob ained
TABLE
2.
Gibbe ellin
p oduc ion
in
he
wild- ype
IMI58289
and
in
ca
mu an s
S ain
Colo ,
da k/ligh
Gibbe -
G ow h
(main
ca o enoid)a
(%)b
(Y)C
IM158289
Albino/O ange
(NX)
100
100
Mu an s
de i ed
om IMI58289
SG2
Albino
(none)
117
97
SG4
Albino
(none)
118
99
SG53
Albino
(none)
112
97
SG54
Albino
(none)
97
92
SG43
Albino
(phy oene)
108
98
SG1
Deep
o ange
(NX)
1
99
SG19
Deep
o ange
(NX)
55
94
SG22
Deep
o ange
(NX)
66
97
SG36
Deep
o ange
(NX)
54
95
SG37
O ange
(NX)
44
95
SG39
Deep
o ange
(NX)
42
98
SG20
Pale
o ange/o ange
(NX)
62
107
SG48
Pale
o ange/o ange
(NX)
51
99
Mu an s
de i ed
om
SG22
SG75
Albino
(none)
45
90
SG76
Albino
(none)
106
97
SG78
Albino
(high
phy oene)
70
98
SG68
Deep
ed
( o ulene)
68
100
SG71
O ange
(NX)
55
98
SG72
Pale
o ange
(NX)
110
97
SG73
Ve y
deep
o ange
(NX)
46
96
a
Acco ding
o
e e ence
3.
Many
s ains
ha e
he
same
colo
a e
g ow h
in
he
ligh
o
in
he
da k.
NX,
neu ospo axan hin.
b
Gibbe ellins
p esen
in
he
cul u e
medium
a e
12
days
o
incuba ion
ela i e
o
hose
o
he
wild- ype
con ols
un
in
pa allel.
The
wild
ype
p oduced
76
±
4
mg
o
gibbe ellins
pe
li e
(mean
and
i s
s anda d
e o
in
18
expe imen s).
c
Glucose
consumed
a e
12
days
o
incuba ion,
ela i e
o
hose
o
he
wild- ype
con ols
un
in
pa allel.
The
medium
ini ially
con ained
80
g
glucose
pe
li e ,
and
he
wild
ype
consumed
31
±
1
g
(mean
±
s anda d
e o
o
17
expe imen s).
The
wild
ype
eached
7.0
±
0.2
g
(d y
weigh )
pe
li e
(10
expe imen s);
he
mu an s
a e aged
6.6
±
0.2
g
(36
expe imen s).
a e
ex ac ion
and
sepa a ion
o
he
gibbe ellins
by
high-
pe o mance
liquid
ch oma og aphy
(8).
Isola ion
o
mu an s
wi h
al e ed
gibbe ellin
biosyn hesis.
An
e en
simple
sc eening
p ocedu e
was
designed
o
he
apid
iden i ica ion
o
s ains
wi h
possible
al e a ions
in
he
p oduc ion
o
gibbe ellins.
This
me hod
was
applied
o
4,100
colonies
g own
om
spo es
ha
had
been
exposed
o
N-me hyl-N'-ni o-N-ni osoguanidine;
34
ga e
educed
o
al e ed
luo escence
in
h ee
successi e
sc eenings.
A
di e -
ence
om
he
wild
ype
was
main ained
by
14
isola es
a e
applica ion
o
he
simpli ied
analy ical
me hod
o
he
p e i-
ous
sec ion.
These
14
isola es
a e
lis ed
in
Table
1.
The
h ee-le e
code
gib
was
chosen
o
he
mu a ions
espon-
sible
o
modi ica ions
in
gibbe ellin
biosyn hesis;
he
new
s ains
ca y
he
alleles
gib-J
h ough
gib-14.
Ca o enoid
con en
o
he
gib
mu an s.
All
o
he
gib
mu an s
excep
SG127
accumula ed
ca o enoids
in
hei
mycelia
a
30°C
in
he
da k
(Table
2).
The
biosyn hesis
o
ca o enoids
was
pho oinducible
in
hese
mu an s
and
in
he
wild
ype:
a
22°C
hey
accumula ed
45
o
111
,ug
o
ca o -
enoids
pe
g
o
d y
weigh
in
he
ligh
bu
less
han
12
,g/g
in
he
da k.
S ain
SG127
was
de oid
o
ca o enoids
unde
all
condi ions
es ed.
These
ca o enoid
analyses
we e
ca ied
ou
wi h
mycelia
g own
on
minimal
DG
aga
(1);
hese
mycelia
p oduced
no
gibbe ellins.
No
co ela ion
was
seen
VOL.
57,
1991
3380
CANDAU
ET
AL.
J.5
LU
U
LU
0
LU
GIBBERELLIC
ACID
(mg
1-1)
FIG.
1.
Fluo escence
(emission
a
464
nm
a e
exci a ion
a
406
nm;
means
+
s anda d
e o s
o
wo
independen
de e mina ions)
a e
applica ion
o
he
simpli ied
analy ical
me hod
o
s anda d
solu ions
o
gibbe ellic
acid.
be ween
he
p oduc ion
o
ca o enoids
and
ha
o
gibbe el-
lins
by
mycelia
g own
in
liquid
medium
(Fig.
2).
The
mu an s
SG133
and
SG134
p oduce
a
mix u e
o
gibbe ellins
ha
is
di e en
om
ha
p oduced
by
he
wild
ype
(7).
The
o he
12
gib
mu an s
p oduce
less
gibbe ellin
han
does
he
wild
ype;
he
subs a es
hus
sa ed
we e
no
di e ed
in o
ca o enoid
p oduc ion
(Fig.
2).
Gibbe ellin
p oduc ion
in
mu an s
de ec i e
o
ca o eno-
genesis.
The
wild
ype
can
mu a e
(3)
o
inc eased
ca o eno-
genesis
(o ange,
deep-o ange,
and
pale-o ange
mu an s
in
he
da k),
o
dec eased
ca o enogenesis
(albino
mu an s
0.3
13$
E
L
0.2
n
a
z
UJ
<
0.1
z
a..
0
0
An
0
10
20
30
40
GGPP
IN
GIBBERELLINS
(jmol
g-1)
FIG.
2.
Gibbe ellin
and
ca o enoid
p oduc ion
by
he
gib
mu-
an s
a e
15
days
o
incuba ion
a
30°C
in
liquid
medium.
To
acili a e
he
compa ison,
he
con en s
o
bo h
g oups
o
e penoids
a e
exp essed
in
e ms
o
he
amoun
(moles
pe
g am
o
mycelial
d y
weigh )
o
he
equi ed
common
p ecu so ,
ge anylge anyl
py ophospha e.
150
CD
E
z
3
ILl
TIME
(days)
FIG.
3.
Gibbe ellin
p oduc ion
by
s ains
IMI58289
(wild
ype),
SG4
(no
ca o enoids),
SG22
(supe p oduce
o
neu ospo axan hin),
and
SG78
(supe p oduce
o
phy oene).
wi hou
ca o enoids),
o
o
he
same
amoun
o
a
di e en
ca o enoid
(phy oene).
The
ca
mu an s
di e
in
he
p oduc-
ion
o
gibbe ellins
(Table
2
ahd
Fig.
3).
All
o
he
inc eases
in
ca o enoid
p oduc ion
we e
accompanied
by
dec eases
in
gibbe ellin
p oduc ion.
Th ee
o
ou
mu an s
ha
lacked
ca o enoids
seemed
o
p oduce
sligh ly
la ge
amoun s
o
gibbe ellins
han
did he
wild
ype.
The
phy oene-accumu-
la ing
mu an
SG43
p oduced
abou
he
same
amoun
o
gibbe ellin
as
did he
wild
ype.
The
cul u e
medium
o
one
o
he
deep-o ange
mu an s,
SG1,
was
p ac ically
de oid
o
gibbe ellins
bu
accumula ed
a
ed
pigmen
wi h
an
unknown
chemical
na u e.
Mu an s
wi h
new
colo
changes
we e
de i ed
(3)
om
one
o
he
deep-o ange
s ains,
SG22.
In
some
cases
he
gibbe -
ellin
p oduc ion
emained
low,
bu
i
e u ned
o
he
wild-
ype
le el
in
wo
mu an s
wi h
dec eased
ca o enoid
p oduc-
ion.
The
a ia ions
in
gibbe ellin
p oduc ion
canno
be
a ib-
u ed
o
di e ences
in
g ow h,
as
shown
by
he
analysis
o
he
emaining
glucose
(Table
1).
The
e y
la ge
a ia ions
in
he
con en
o
gibbe ellins
and
ca o enoids
in
di e en
s ains
app oxima ely
compensa e
each
o he .
In
he
s ains
gi en
in
Fig.
4,
he
a ia ion
coe icien s
a e
109%
o
he
ca o enoid
con en s
and
only
12%
o
he
sum
o
bo h
g oups
o
e penoids.
E ec
on
gibbe ellin
p oduc ion
o
agen s
ha
a ec
ca o-
enogenesis.
Ligh
had
no
e ec
on
he
p oduc ion
o
gibbe -
ellins.
This
is
shown
in
Fig.
5
o
he
wild
ype
unde
condi ions
ha
no mally
allow
o
ep ess
gibbe ellin
p oduc-
ion
and
o
s ain
SG4,
a
whi e
mu an
unable
o
make
ca o enoids
ei he
in
he
ligh
o
in
he
da k.
Unde
he
same
condi ions
he e
was
no
pho oinduc ion
o
ca o enogenesis:
a e
15
days
in
liquid
medium
in
he
ligh
o
in
he
da k,
he
wild- ype
mycelia
con ained
a
o al
o
24
o
29
,ug
o
ca o enoids
pe
g
o
d y
weigh .
The
wild
ype
does
no
lend
i sel
o
he
s udy
o
inhibi o s
o
ca o enogenesis
because
o
i s
low
ca o enoid
con en .
The
deep-o ange
s ain
SG22
had
been
used
p e iously
in
i s
place
(3).
a-Picoline
inhibi s
lycopene
cycliza ion
and
delays
136
135
122
.
129
134
139
0
IM1I58289
*
128
0
0
121
*
133
0
124
.137
127
123
-.
.d&.
I
APPL.
ENVIRON.
MICROBIOL.
GIBBERELLINS
AND
CAROTENOIDS
IN
GIBBERELLA
MUTANTS
3381
40-
E
%.
0.
0
30
20
-
10
-
0-
IMI
l58289
SG4
SG22
STRAIN
FIG.
4.
P oduc ion
o
gibbe ellins
(=)
and
ca o enoids
(
)
by
wild- ype
IM158289
and
some
ca o enoid
mu an s
a e
15
days
o
incuba ion
a
30°C
in
liquid
medium.
The
con en s
o
bo h
g oups
o
e penoids
a e
exp essed
in
e ms
o
he
amoun
(moles
pe
g am
o
mycelial
d y
weigh )
o
he
equi ed
common
p ecu so ,
ge anylge-
anyl
py ophospha e.
g ow h
in
G.
ujiku oi.
x-Picoline
comple ely
blocked
gib-
be ellin
p oduc ion
when
added
a
a
concen a ion
o
2
g
li e -'
ei he
be o e
he
inocula ion
o
he
cul u es
(Fig.
6)
o
a e
gibbe ellin
p oduc ion
had
s a ed
(Fig.
7).
Diphenylamine
(20.3
mg
li e -')
and
,-ionone
(200
mg
li e -')
inhibi ed
g ow h
when
added
be o e
inocula ion
o
he
cul u es.
When
hey
we e
added
o
9-day-old
cul-
u es,
he e
was
a
pa ial
inhibi ion
o
gibbe ellin
accu-
1501
I
cn
z
i
Lu
cm
100
50
TIME
(days)
FIG.
5.
E ec
o
ligh
on
gibbe ellin
p oduc ion.
Symbols:
0,
O,
and
A,
cul u es
g own
in
he
ligh ;
0,
*,
and
A,
cul u es
g own
in
he
da k;
Au
and
A,
s ain
SG4
g own
in
liquid
medium;
0
and
0,
wild- ype
IM158289
g own
in
liquid
medium;
O
and
*,
wild
ype
g own
in
liquid
medium
wi h
4.8
g
o
NH4NO3
pe
li e .
6
LU
0
ao
0
LU
20
120
100
ab80
60
-I
LU
co'
40
20
20
IT
5
10
15
20
25
TIME
(days)
FIG.
6.
E ec
o
a-picoline
on
he
mycelial
d y
weigh
(A)
o
s ain
SG22
and
on
he
glucose
(B)
and
gibbe ellin
(C)
con en s
o
he
medium.
Symbols:
0,
a-picoline
(2
g
li e -')
added
o
he
medium
be o e
inocula ion;
0,
cul u es
wi hou
oL-picoline.
mula ion
(Fig.
7),
which
was
p obably
nonspeci ic
because
i
was
accompanied
by
a
simila
inhibi ion
o
g ow h.
The
conse ed
luo escence
spec a
indica ed
ha
hese
chemi-
cals
p oduced
no
g oss
changes
in
he
mix u e
o
gibbe el-
lins.
VOL.
57,
1991
-Vj
3382
CANDAU
ET
AL.
Ox
100
E
z
LU
s5
10
1S
20
25
TIME
(days)
FIG.
7.
E ec
on
gibbe ellin
p oduc ion
by
SG22
mycelia
o
a ious
chemicals
added
o
9-day-old
cul u es
(a ow).
Symbols:
0,
cul u es
wi hou
chemical
addi i es;
A,
emulsi ie s
e hanol
and
Tween-80;
*,
a-picoline;
*,
diphenylamine;
A,
13-ionone.
DISCUSSION
New
me hods.
The
isola ion
o
he
gibbe ellin
mu an s
and
he
o he
esul s
in
his
pape
would
ha dly
ha e
been
possible
wi hou
he
de elopmen
o
wo
new
me hods.
The
luo escence
o
he
eac ion
p oduc s
o
gibbe ellins
wi h
sul u ic
acid
is
he
ounda ion
o
adi ional
analy ical
me h-
ods
(14).
Ou
p ocedu e
a oids
he
ex ac ion
and
pu i ica-
ion
s eps
wi h
hei
concomi an
losses
o
gibbe ellins.
The
p ocedu e
migh
gi e
alse-posi i e
eac ions
wi h
chemicals
o he
han
he
gibbe ellins.
Tha
his
is
no
he
case
o
ou
Gibbe ella
s ains
is
shown
by
he
negligible
luo escence
obse ed
wi h
he
wild
ype
in
he
p esence
o
a-picoline
and
wi h
mu an s
de oid
o
gibbe ellins.
Ou
apid
sc eening
p ocedu e
may
be
used
no
only
in
he
sea ch
o
mu an s
bu
in
o he
expe imen s
as
a
apid
i s
es
o
iden i y
he
samples
ha
dese e
p ecise
analysis.
Rela ionship
be ween
he
biosyn heses
o
gibbe ellins
and
ca o enoids.
S ain
SG127
p oduced
negligible
amoun s
o
bo h
e penoids.
A
single
mu a ion
could
block
he
ans e
o
a
p enyl
esidue
o
amesyl
py ophospha e
o
p oduce
ge anylge anyl
py ophospha e
o
shu
o
a
possible
common
egula ion
o
bo h
pa hways.
The
mu a ional
incapaci y
o
p oduce
amesyl
py ophospha e
should
be
le hal
o
lack
o
s e ols.
The
inc eased
ca o enogenesis
obse ed
in
he
deep-
o ange
mu an s
is
quan i a i ely
compensa ed
by
a
de-
c eased
p oduc ion
o
gibbe ellins.
The
same
seems
o
be
he
case,
in
he
opposi e
di ec ion,
o
he
albino
mu an s
de oid
o
ca o enoids.
These
mu a ions
do
no
a y
he
supply
o
p ecu so s
bu
di e
hem
o
he
gibbe ellin
o
he
ca o -
enoid
b anch.
The
di e sion
may
occu
a
he
le el
o
ge anylge anyl
py ophospha e
o
any
p e ious
p ecu so .
The
albino
mu an s
may
be
unable
o
syn hesize
phy oene
om
ge anylge anyl
py ophospha e.
The
compensa ion
ule
does
no
apply
o
o he
mu a ions,
which
seem
o
a ec
only
one
o
he
pa hways.
This
is
he
case
pa icula ly
wi h
mu a ions
ha
block
he
p oduc ion
o
gibbe ellins.
A
di e sion
o
he
gibbe ellin
p ecu so s
in o
he
ca o enoid
pa hway
would
esul
in
imp essi e
pigmen-
a ion
le els
(abou
10
mg
g
o
d y
weigh -').
Whi e
illumina ion
does
no
a ec
he
p oduc ion
o
gib-
be ellin
and
ca o enoids
in
ou
liquid
cul u es.
The
ligh
in ensi y
used
is
no
e y
high
bu
is
enough
o
b ing
abou
conside able
pho oinduc ion
o
ca o enogenesis
on
aga
cul-
u es.
a-Picoline
blocks
he
biosyn heses
o
bo h
neu ospo a-
xan hin
(2)
and
gibbe ellins
in
Gibbe ella.
I
does
no
p e en
he
p oduc ion
o
he
common
p ecu so
ge anylge anyl
py ophospha e,
since
neu ospo axan hin
is
eplaced
by
ly-
copene.
Thus,
a-picoline
inhibi s
he
o ma ion
o
he
I8
ing
o
he
ca o enoids
and
is
likely
o
inhibi
he
cycliza ions
in
he
gibbe ellin
pa hway.
Bo h
pa hways
include
dehyd ogena ion
s eps.
Diphenyl-
amine
and
,-ionone
inhibi
he
dehyd ogena ions
om
phy-
oene
o
o ulene
bu
do
no
seem
o
ha e
any
speci ic
e ec
on
gibbe ellin
biosyn hesis.
The
h ee
chemical
inhibi o s
s imula e
he
consump ion
o
glucose
bu
inhibi
g ow h.
They
appea
o
uncouple
he
ca abolism
o
glucose
om
i s
inco po a ion
in o
ungal
biomass.
ACKNOWLEDGMENTS
We
app ecia e
he
echnical
assis ance
o
M.
C.
Vale o
Qui 6s
and
J.
C6 doba
L6pez
and
he
sugges ions
and
commen s
o
Edua do
R.
Beja ano
and
Pe e
M.
B amley.
We
app ecia e
he
inancial
suppo
o
he
Eu opean
Commission
(g an
BAP-0395-E)
and
Comisi6n
In e minis e ial
de
In es igaci6n
Cien i ica
y
Tecnica
(g an
BT87-34).
REFERENCES
1.
A alos,
J.,
J.
Casadesus,
and
E.
Ce da-Olmedo.
1985.
Gibbe ella
ujiku oi
mu an s
ob ained
wi h
UV
adia ion
and
N-me hyl-N'-
ni o-N-ni osoguanidine.
Appl.
En i on.
Mic obiol.
49:187-
191.
2.
A alos,
J.,
and
E.
Ce dai-Olmedo.
1986.
Chemical
modi ica ion
o
ca o enogenesis
in
Gibbe ella
ujiku oi.
Phy ochemis y
25:
1837-1841.
3.
A alos,
J.,
and
E.
Ce da-Olmedo.
1987.
Ca o enoid
mu an s
o
Gibbe ella
ujiku oi.
Cu .
Gene .
11:505-511.
4.
A alos,
J.,
and
E.
Sch o .
1990.
Pho oinduc ion
o
ca o enoid
biosyn hesis
in
Gibbe ella
ujiku oi.
FEMS
Mic obiol.
Le .
66:295-298.
5.
Bea de ,
J.
R.
1983.
In
i o
di e penoid
biosyn hesis
in
Gibbe -
ella
ujiku oi:
he
pa hway
a e
en -kau ene,
p.
251-387.
In
A.
C ozie
(ed.),
The
biochemis y
and
physiology
o
gibbe ellins,
ol.
1.
P aege
Publica ions,
New
Yo k.
6.
B iickne ,
B.,
D.
Blechschmid ,
G.
Sembdne ,
and
G.
Schneide .
1989.
Fungal
gibbe ellin
p oduc ion,
p.
383-429.
In
E.
J.
Van-
damme
(ed.),
Bio echnology
o
i amins,
pigmen s
and
g ow h
ac o s.
Else ie
Science
Publishe s,
L d.,
London.
7.
Candau,
R.
1991.
P oducci6n
de
gibe elinas
po
Gibbe ella
ujiku oi.
Ph.
D.
hesis,
Uni e sidad
de
Se illa,
Se ille,
Spain.
8.
Candau,
R.,
and
P.
B amley.
Unpublished
da a.
9.
Dubois,
M.,
K.
A.
Gilies,
J.
K.
Hamil on,
P.
A.
Rebe s,
and
F.
S.
Smi h.
1951.
A
colo ime ic
me hod
o
he
de e mina ion
o
suga s.
Na u e
(London)
168:167.
10.
Geissman,
T.
A.,
A.
J.
Ve bisca ,
B.
0.
Phinney,
and
G.
C agg.
1966.
S udies
on
he
biosyn hesis
o
gibbe ellins
om
(-)-
kau enoic
acid
in
cul u es
o
Gibbe ella
ujiku oi.
Phy ochem-
is y
5:933-947.
11.
Goodwin,
T.
W.
1980.
The
biochemis y
o
he
ca o enoids,
ol.
1,
2nd
ed.
Chapman
and
Hall,
London.
12.
Je e ys,
E.
G.
1973.
The
gibbe ellin
e men a ion.
Ad .
Appl.
Mic obiol.
13:283-315.
13.
Pha is,
R.
P.,
and
R.
W.
King.
1985.
Gibbe ellins
and
ep oduc-
i e
de elopmen
in
seed
plan s.
Annu.
Re .
Plan
Physiol.
36:517-568.
14.
Takahashi,
N.,
I.
Yamaguchi,
and
H.
Yamane.
1986.
Gibbe el-
lins,
p.
57-151.
In
N.
Takahashi
(ed.),
Chemis y
o
plan
ho mones.
CRC
P ess,
Boca
Ra on,
Fla.
APPL.
ENVIRON.
MICROBIOL.