P l antMolecular

B io lo g y2
Editedby

R. G. Herrmann
and

B.A. Larkins
NATOASlSeries

Vol.212
SeriesA: LifeSciences

Proceedings of a NATO AdvancedStudy Institute

on Plant MolecularBiology,
held May 14-23, 1990,
in Elmau,Bavaria,Germany

Llbnar y

of Congr es s C .tr l ogl ng- l n- Publ

I{ ATO Advanced Study

Schloss ElDau)
Plant iolecula'
Lar k I nS.

lnst r tute
btology

on Pl anr ttol ec ul r r

2 / edi ted

l c .tl on

D
t"l

D at,

Bl ol ogy

by R .G. H e...ann

( 6th

: 1990

and B, A.

Pr oceedlngs o. the Sr r th N AT O Adv anc ed Study Ins t l tute on Pl ant

Molecular Er ology held H ay 14- 23, 1990, tn El r au, Bav ar ta,0er r any .
If,cludes br blr og.aphlcal
.efer enc es and r nder .
ISaN 0- 306- 44924- 5
1. Plant r ole:ular
br ol ogy - - C ongr es s es .
I. H er r tr ann, R . G.
( Rer nhold G. )
T i r l e.
:I.
Laek r ns - 8. A. ( B.1r n A.)
l II.
oK728.N38
t990
581.8- - dc20
I I -26944

tsBN G306440245
O 1991 PlenumPress,New York
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FLORAL HOMOEOTICAND PIGMENTMUTATIONSPRODUCED

BY TRANSPOSON-MUTAGENESIS
IN Antirrhinum

majus

Ros em ar y Car penc e r , S a n d r a D o y l e , D a L u o , J u s t i n G o o d r i c h ,

J 6s e M . Roner o, R o b e r c E l t i o t ,
Ruth Hagrath and Enrico Coen
Genecics Departxnent, John Innes Institute,
John Innes Cencre
for Planc Research, Colney l^ane, Norwich NR4 7lJH, U.K.

INTRODUCTION
M a n y h o m o e o tic
genes
affecE i ng
fl ow er
norphogenesi s
have
been
d e s cr ib e d
in d ive r se
sp e cie s,
al chough
there
have been few atteupts
to
th e se
nay to the nechani sn
relaEe
in a syste n a cic
of fl oral
devel opmenc
( H e ye r o wicz
e t a 7 .,
1989) .
In L742 i c vas the pel ori c
forrn of Li nari a
v u l g a r is
ch a c m a d e L in n a e u s
change hi s ni nd about the fi xi ty
of speci es
( L i n na e u s,
L744)
a n d o ve r
a cencury
D arni n
descri bed,
l ater
and w as
(D arw i n,
i n E rig u e d
b y, ch e b e h a vio u r
o f the pel ori c
A nti rrhi nun
We
1868).
h a v e u se d Er a n sp o so n - n u ta g e n e sis
to tenerace fl oral
mutati ons
honoeoti c
in
wiCh a vie w to stu dyi ng
A n t ir r h in u m
and i sol ati ng
the genes i nvol w ed.
One
o f th is
a d v an ta g e
a p p r o a ch is thac cransposon
i ntegraci on
can be used E o
g e n e s a n d su b se q u e n t
i d e ncify
exci si on
can be used to prove
C hat che
(H arti n
c o r r ect
g e n e h a s b e e n iso la ted
et
a)-.,
In addi ci on,
f985).
e xcisio n
i n p r e cise
ca n g e n e rate
w i th
al l el es
al -cered
gene expressi on
( A l m e id a
e c a l.,
1989).
An tir r h i.n u m
m a ju s p r o vides
i deal
experi nencal
rnaceri al
for
thi s
a p p r o a ch .
T h r e e d iffe r e n t
cr a nsposons have been i sol ated
from i c (C oen ec
al.,
1 9 8 9 ) a n d th e ce ch n iq u e
of transposon-taggi ng
has been successful l y
( Ha r cin
used
ec aL , 1985).
S everal
genes encodi ng
enz)rmes i n
rhe
p ig m e n t
a n t h o cya n in
pathw ay have been i sol ated
b io syn th e cic
and provi de
good
m a r ke r s
fo r
n o n ito r in g
C ransposi ti on
and
the
C rappi ng
of
nerJ
t r a n sp o so n s.
T h e r e is a lso a good geneE i c nap w i ch rnany w el l -characteri sed
m u ca cio n s
a ffe ctin g
(S rubbe,
flo we r
d evel opnent
1955).
In the w i l d-type
o f A n tir r h in u m
m a ju s th e flo we rs
are borne i n a spi ral
up the sten,
forni ng
a n in flo r e sce n ce .
Ea ch flo wer
grow s i n the axi l
of
a bract
and i s
z y g o m o r p h ic.
In
Er a n sve r se
secti on,
E he fl over
rnay be consi dered
as
c o mp r isin g
fo u r
co n ce n tr ic
r ings or w horl s,
each contai ni ng
several
organ
m e mb e r s wh ich
will
b e r e fe r r e d
to as upper or l ow er dependi ng
on chei r
p o s itio n
r e la cive
co th e b r a ct rehi ch i s consi dered
the l ow est organ.
Ihe
f i r s ts
o r o u ce r m o sE wh o r l co n p r i ses
fi ve sepal s,
the l ow esc bei ng al cernace
t o th e b r a cc.
T h e se co n d wh o r l consi scs of fl ve
petal s
uni ted
for part
of
c h e ir
le n g th
co fo r m th e co r o l l a
tube vi th
fi ve
l obes.
Fi ve st:unens are
a n d co n sticu te
i n i c ia ce d
th e thi rd
shorl ;
how ever,
the upperrnost scaoen
r e r n a in s ve stig ia l
so ch a t th e ful ry
devel oped fl ow er has onl y four scamens,
the upper
tvo
b e in g
sh o r te r
than the l oner
tw o.
A bi l ocul ar
ovary
c o n stitu Ee s
th e fo u r th
wh o r I.
The i denti ty
r,ri l I be i ndi caced
of the vhorl s
PIut Molccular Biology a Editd by R.G. Hcrrmann md
^' B. trkiro, Plcnun! hcss, New Yuk l9l

537

!;;

.-";,$''{.'rtfil $'
'F* 1i' SSti?,"
'.*.r:

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ti

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F lg .
l.
wild - cyp e

by
o f cycToi dea
l sol ated
Hu ca tio n s
p r o g e n ito r
is show n at the cop.

in sequence scarEing fron

petal , sta.Een, carpel .

che first

whorl

E ransposon-E utatenesi s.

so that

uild-type

is

sepal,

h
.,

IS
TR.L\SPOSONMUTAGENES
active
I n eac h c as e, 10 o r 1 5 p l a n c s o f l i n e s c a r r y i n g
highly
transposons were gror.rn at a conscant 15"C, the cenperature
ac which che
gr eat es c f r equenc y of c r a n s p o s i t i o n o c c u r s ( H a r r i s o n a n d F i n c h a n , 1 9 6 4 ;
and seed
Har r is on and Car pent er , 19 7 3 ) . I h e s e p l a n t s w e r e s e l f - p o l l i n a t e d
capsules vere collected separacely.
Seed from these plants gave rise to
of eit her L5 or 3 0 p l a n t s f r o r n e a c h c a p s u l e , w i t h a t o t a l . o f
f anilies
13,000 H, plants grown in the glasshouse.
As nost nutations are likeIy to
be recessive many of these plants should have been heterozygotes; therefore
t hes e H, plant s ner e als o s e l f - p o l l i n a t e d .
For the H, generation, seed
(depending on
fron each fanily was sotn to give either 48 or 96 plants
whecher the H, fanily
cornprised 15 or 30 plants)
to give a cotal Hz
generation of 40,000 plancs grown in the field.
Seweral flower honoeocic
s elec t ed f or analy s is .

538

and pignent

mucacions were obtained

and

,l

flt
N

fl

Floral

h o m o e o tic

n u ta n ts

The floral
honoeoCic Eutations that were lsolated can be divided into
The first class affects the identity
four classes.
of menbers wichin the
same whorl and includes Ehe c/cloidea
alleles
rshich confer varying degrees
synroetry to the florrer.
of radial
Two independent Dutations givlng this
phenot)rpe sere obtained (Fig. l) and genetic analysls of these allowed Eno
groups of cycToidea alleles
to be defined.
Extrene alleles of one group
confer a pelorie phenotype in nhich all uembers of a wtrorl resenble the
Iowesc nember of the wild-cype flower.
The second group also give flor.rers
which are more synnetrical
chan wild-type
buc retain
a degree of
zygomorphy. Crosses betseen plants carrying alleles
fron the groups give
F, plants with an alnost wild-type phenotype buc wiCh Eno snall notches on
the lower flower lip, shoving ChaC alleles
fron the tiro groups partiaLly
conplenent each other. This suggests Chat cycToidea nay be a complex locus
composed of cwo interacting
funccional cornponents. The second class of
homoeotic mutations affecE the identity
of che rrhorls.
one Eutsant of this
class is ovuilata which, unlike the other nutations,
sas deCected in the H,
generation, indicating chat it was dominant or semi-dominant to rrild-type.
Several differenc
Hr plants,
all derived fron the same parent, showed a
range of related phenotypes. The nost severe of these ras self-pollinared
and gave an H2 of 3 wild-type,
12 parencal phenotypes and 6 Eutants uich
a new excrene phenotype. The firsc whorl of thls extreme phenocype (Fig.
2) conprised five carpels, the uppernost lacking ovules and the four lorer
being uniced to give an ovary
four owule-bearing rocuri terninating
'ich
in a uniced band of stylar cissue.
The second whorl had three lower sca!ns
whire
the upper trro nembers remained as snall
vestigial
or aborced
structures;
the chird and fourth nhorls rrere wild-cype.
Ihe near 1:2:1
segregation of the l{r progeny also suggested that the oyulata nucation nas
seni-doninant,
the parental phenoc)pe being thac of the heterozygote and
che extreme phenotype being the ovurata honozygote. This was confirnec in
the next generation.
Other mutacions of this second class were also idencified.
ltutacions
at cwo unrinked roci, deficiens and separoidea, uere obtained which. in che
most excreme fonn, resurted in sepals growing in prace of petals and
carpeLs inscead of stamens to give the phenocype sepal, sepal, carpel.
Ihe
uhorl did not normally
fourth
develop in che extreoe phenotype but
conprised the nornal bilaterar
ovary of wild-type in Ehe less severe foras.
A chird unlinked locus, grobosa, has arso been descrlbed which gives this
phenocJrpe (Kuckuck and Schick, 1930).
The deficiens allere was somatically very unstable.
Dlscrece clonar
pacches of pipented
petal tissue,
sonetimes couprising onry four celrs
were observed in the second ron of sepals (Fig. 3a and b).
occasionally
entsire wird-type flowers were produced on che mutant, presumably causec by
early sonatic reversion events.
rn addition,
snalr frowers contaicin!
much-reduced stamens nere someciDes seen and reserobled che phenocype oi
deficiensnicotlnoides (Hercvig, Lgz6).
The phenoc)rpe of sepaloidea was arso
wery wariable sith a tendency to produce large areas of petaloid
tissue,
usually edged vich sepal-like
areas in the second whorr (Fig. 3 c and d).
The chird class of homoeotic genes affect
both the identicy
and
number of whorls.
This includes che pleniflora
Eutants vhich give florers
uich the firsc chree nhorls of che type separ, petal, pecal.
rhe fourch
whorl couprises Ewosepaloid/carpeloid/petaloid
structures and nichin these
a p rollf er ac ion
of pet aloid wh o r l s o c c u r s .
Tvo of the nutations uere unstabre sonaticarly
and gave occasional
vild-type
flowers on oEherr,rise Bucanc spikes.
seed fron ihese gave boch
vild-cype and nucanr progeny. The wird-rype progeny were self-pJrri.,.c"d

539

?i,
rl i.

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.;.t
t:*

','r.

it{.

4.1

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'.isi',

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'i :

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tdi.
;

tl

'-l.,ll
',*':j{{,

.4 t a .

r4

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L it ,:4
i ,b."--$
7.4

1',t
>.f

}. ;,i
;l-*
,I

-*l'

lrl

*ti
.t

{
..:[
'a

:
t
6

..'t
r :l
;1.{

i,Z
{
'lt

_z
F lg .

2.

In flo r e scence

of

the

extreme

ovul aca

nutati on.

and in each case segregated for mucants and wild-t)ryes.

These results
indicaced chac che mutations were recessive and could reverc to nild-type
in both soEatic and germinal cissue.
However, ic has noE yec been
established
if
these
mutacions
belong
to
the
s:ilre or
different
conpleoencation groups .

fourth

The potential
for indeterninace gronth is further illustraced
by che
class of nuEants nhere the switch from vegetaEive grorrth co thac of

540

Ftg.
3'
Diversity
of flowers produced by deficiens
and separoidea
muEacions. Top row: deficiens flowers showing che second whorl of sepals
and che united five carpels of the third whorl.
Flower b shows a clonal
pa t c h of pet ar c er ls on t he s e c o n d w h o r l o f s e p a r s a n d a c e n c r a l c a r p e l .
Bottom row: flosers fron che sepaloidea mutation which che mosc extreme
forn on che lefc.

che reproductive spike nas as nornal.

However, in the floricaula
Dutant,
instead of a flor.rer being produced in the axil of each bract,
a secondary
shooc was forned.
This shooc in turn produced bracts sithin
che axils of
which further
shoo.s nre produced.
This
process
courd concinue
indefinitely
to forn an indeteruinate shoot shich has lost it.s abirity
to
flower (Fig. 4).
The wird-type froricaura produc. is cherefore required
for
swicching
indeterninate
shooc neristens
to floral
neriscems and
pres'mabry activates expression of the other classes
of floral- honoeotic
g en es eit her dir ec t ly
or indir e c t l y .
Ihe mutation was propagaced vegetacively
and a nunber of cuccings
lrere groerr at 150c. one of chese produced occasional
frowers vhich uere
self-pollinaced
and five capsures of viable
seed were obcained.
The
progeny from four of chese were all eutanc;
however one capsule gawe
progeny which segregated to give 4 Dutanc
and 1g wild_type
plancs,
indicating
that gerrninal reversion to the r.rild-type allele
had occurred.

541

II

$
l

PL g .

4.

Sp ike

of

th e

flo r i caul a

nutatl on

shorl ng

Lndeterui nate

groerth.

Ihe progeny fron the wild-types

shoved that some gere heterozygous
and
ochers homozygous for the wild-t)rpe allele.
Further revertant progeny fron
a number of independent events have norr been obtained.
Hucant phenotypes
have been described
in Arabidopsis which show nany si-oilarities
to those
(Haughn and Somerville, 1988). The remarkable sinilarities
of,{ncirrhinum
between mucants in Antirrhinua
which belong
and Arabidopsis,
to two
taxonomically
distanc planc subclasses, sutgest that the genetlc conErol
of whorl
conserved
ldentity
has been highly
the
evolution
in
of
dicocyledonous plants.
Cell

autonomy of nutants

Many of che homoeocic rnutants show lnscability

in somatic or gerninal
Eissue, suggesting chac they nere transposon-induced.
In sone cases, the
instability
nay be used to investigate cell-autonouy of the genes affected.
The deficiens-627
allele
gives clonal patches of petal
tissue
on the
of the second whorl of sepals.
epidernis
These patches have very sharp
boundaries and nay be explained by sonattc excision of a transposon fron
che deficiens
locus restoring
gene function.
This suggests that the
product of the vild-cype deficiens gene is not diffusible
between cells and
acts ce1l-autononously,
at least in the epidernis.
The observation of some
very
patches
small
indicates
that
product
the deficiens
is
acclve
throughout
development of the petal, fron the early stages when petal and
sepal pri,nordia become distinct
to the final cell divisi-ons of the petal.
Honever, the consequences of deficiens expression nay be different
at each

542

pacterns
of
stage so that at early stages 1c rnight affect
developmental
whereas ac
and expansion and hence the forn of the petal,
cell division
later scages ic nay affect cell face.
Since the clonal patches of the deficiens'621 allele occur only ln
whorl of sepals, there Eust be other genes
the second and not the first
in the second nhorl throughout its development and
that act specifically
that are necessary either fot deficiens expression or for the actlon of che
since
producc.
Two such genes nay be gTobosa and sepaloidea,
deficiens
The
mucation of these gives a siruilar phenotype to che deficiens nutant.
which gives a sinilar
nutaEion of Arabidopsis,
apetala-3
PhenotJpe to
is also thought co acc uP co a late stage of organ developments
deficiens,
on the basis of texnperacure shifc experiuents (Bownan et al. 1989).
Transposon-tagginE
.

and Erapping

were
obcalned
and
pigrlencacion
mucancs irere
also
Severa{
Flve ouc of
to determine if they were transposon induced.
characterlsed
diagnostic
of
lnstabilicy,
six mucants showed somaEic and gerninal
cloned
One muCacion, niv-600, was in a previously
Cransposon insercions.
in the
gene and molecular analysis showed that lc was caused by insertion
exon of a new transposon, Tan4, which is present ln about 10 copies
first
in the parental line.
revercancs
of incoloraca-601 and its gerninal
Molecular analysis
of Tanl,
using several Tan probes, shoned that it was due co insertion
ac deLiTa, a recessive regulacory gene thaE
whilst an unstable nutation
of a Tam2
tube. was the result
blocks pignentaEion
in the corolla
which gives leaves with dark green spots
The olive-505 allele,
insertion.
in a yellou background, showed several features typical of Tan3 lnsertions.
The frequency of spocs was uuch greater shen the Dutant rtas trown aE. 15oC
and
coopared co 25"C, as has been found for Tan3 lnsertions ac the pallida
1989) .
Furtheruore, when olive-605 nas crossed
n ive a loc i ( Coen ec al. ,
ScabiTiser, a gene known specifically
to tnhl.bic Tan3
co a line carrying
excision ( Car pent er ec aI . , 19 8 7 ) , t h e F 2 s e g r e g a t e d p l a n c s s h o w i n g v e r y
Ihese plants failed Eo trou to Daturicy,
few if any dark treen spots.
presunably because of photosynthetic
deficiency,
indicating
chac the
via bi lic y
of oliv e- 605 depends o n c r a n s p o s o n e x c i s i o n .
Molecular analysis
of olive-605 and its revertancs confirned that the nutation was caused by
and has led to isolation
Tan3 insertion
of the ol.ive locus.
This is che
pigpentation
Eime a yellow
firsc
leaf
Eucant has been characcerised
molecularly and chis should now allow a nes approach !o the study of such
te ne s .
The resulls show thac uost of the nenly produced pitpent nutacions
were caused by transposon insertion and that lt is possible to isolaCe the
affected gene using cloned transposons as cags. Ac leasc four differenc
Cransposons are active in the lines used for ruEagenesis one of shich,
Tam4, was newly isolated
by crapping it in a previously cloned gene. As
furEher genes and nucations are characterised iC should be possible to trap
Dore transposons and so define che full range accive in che lines.
These
could be used to decernine which transposons nere responsible
for che
floral
honoeotic nuEations and so allow the isolacion
and oolecular
analysis of the genes involved.
Acknowl edgements
l.Ie thank David Hopwood, Cathie l{artin and l{ichal Goldsctrnidt-Clermont
for helpful discussions and Kathqm Blewett, Karen Ingle and Audrey Cooper
for strmmer vacational
asslstance.
We are grateful
to Hans Somer for
generously providing
us wich the sequence and probes of Tanl prior
co

543

publicacion,
Peter scott and Andrew Davies for photography, the Glasshouse
and Field services Departmenc for growing the plants and Anne llillians
for
cyping Ehe nanuscript.
I.le acknowledge a generous grant froo the Gatsby Foundation
enabled us to carry out Ehe transposon-Dutagenesis experiment.

which

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Alm eida, J . , Car pent er , R . , R o b b i n s , T . P . , H a r r i n , C . , a n d C o e n , E . S . ,
1989, G enet ic in t e r a c t i o n s
underlying
flower
color
patterns
in
k t t ir r hinum
najus , G e n e s a n d D e v . , 3 : 1 7 5 8 .
Bownan, J . L. , Sny t h, D . R . , a n d [ e y e r o w i t z ,
E. H., 1989, Genes direccing
flower developoent ia Arabidopsis, The planc Cell,
l:37
Car pent er , R. , M ar t in, C . R . , a n d C o e n , E . S . , 1 9 8 7 , C o m p a r i s o n o f g e n e t i c
.behaviour of the transposabre elenent Tan 3 at two unrinked pigmenc
I oc i in Anc ir r hinu a n a j u s , 1 1 o 7 .G e n . G e n e t . , 2 O l : 8 2 .
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Har r is on,
B. J . ,
and C a r p e n t e r , R . ,
1973,
A conparison
of
the
instabilities
at the nivea and parlida loci in Antirrhinum majus,
Her edit y , 3l: 309.
Har r is on, B. J . , and Fin c h a r n , J . R . s . , 1 9 6 4 , r n s r a b i l i r y
ac che par locus
in ,4acirrhinum majus. I) Effects of environment on frequencies of
somatic and geruinal nutation,
Heredity,
19:.237.
Haughn, G . W . , and S o n e r v i l l e ,
C. R.,
1988, Genetic
conrrol
of
nor phogenes is in A r a b i d o p s i s , D e v . G e n e t . , 9 : 7 3 .
Her t wig,
P. , 1926, Ein n e u e r F a r r v o n n u l c i p l e n
Allelomorphisms
bei
Ant ir r hinun,
Z. f . i n d u k t A b s c . - u . V e r e r b u n g s T . , 4 L : 4 2 .
Kuc k uc k , H. . v on, and S c h i c k , R . , 1 9 3 0 , D i e E r b f a k r o r e n b e i A n t i r r h i n u m
oajus und ihr e B e z e i c h u n g . Z . t .
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