D EVELO PM EN T AL BIO LO G Y 202, 136 –151 (1998)
ART IC LE N O . D B988980
Identification of Gam etophytic Mutations Affecting
Fem ale Gam etophyte D evelopm ent in A rab id op sis
Cory A. Christensen, Santhi Subram anian, and Gary N . D rew s 1
D epart m en t of Biology , U n iv ersit y of U t ah , 257 Sou t h 1400 E, Salt Lak e C it y , U t ah 84112-0840
The fem ale gam etophyte (em bryo sac or m egagam etophyte) plays a critical role in sexual reproduction of angiosperm s. It is
the structure that produces the egg cell and central cell w hich, follow ing fertilization, give rise to the seed’s em bryo and
endosperm , respectively. In addition, the fem ale gam etophyte m ediates a host of reproductive processes including pollen
tube guidance, fertilization, and the induction of seed developm ent. Several m ajor events occur during m egagam etogenesis,
including syncitial nuclear divisions, cellularization, nuclear m igration and fusion, and cell death. While these events have
been described m orphologically, the m olecules regulating them in the fem ale gam etophyte are largely unknow n. We discuss
a genetic screen based on reduced seed set and segregation distortion to identify m utations affecting m egagam etogenesis and
fem ale gam etophyte function. We report on the isolation of four m utants (fem 1, fem 2, fem 3, and fem 4) and show that the
four m utations m ap to different locations w ithin the genom e. Additionally, w e show that the fem 1 and fem 2 m utations
affect only the fem ale gam etophyte, w hile the fem 3 and fem 4 m utations affect both the fem ale and m ale gam etophyte. We
analyzed fem ale gam etophyte developm ent in these four m utants as w ell as in the gfa2, gfa3, gfa4, gfa5, and gfa7 m utants.
We found that the fem 2, fem 3, gfa4, and gfa5 m utants abort developm ent at the one-nucleate stage, w hile the fem 1, fem 4,
gfa2, gfa3, and gfa7 m utants are affected in processes later in developm ent such as polar nuclei fusion and cellularization.
The establishm ent of a genetic screen to identify m utants and the developm ent of a rapid procedure for analyzing m utant
phenotypes represent a first step in the isolation of m olecules that regulate fem ale gam etophyte developm ent and
function. © 1998 Academ ic Press
K ey W ord s: fem ale gam etophyte developm ent; A rab id op sis; nuclear fusion; cellularization; plant reproduction;
em bryo sac.
IN TROD U CTION
Plan t s h ave a t w o-st aged life cycle in w h ich a h aploid
gam et oph yt e gen erat ion alt ern at es w it h a diploid sporoph yt e gen erat ion . An giosperm s are h et erosporou s an d h ave
m ale an d fem ale gam et oph yt es. T h e m ale gam et oph yt e
(pollen grain ) develops w it h in t h e st am en ’s an t h er an d
con sist s of t w o sperm cells en cased w it h in a veget at ive cell.
T h e fem ale gam et oph yt e (em bryo sac or m egagam et oph yt e)
develops w it h in t h e carpel’s ovary an d exh ibit s a variet y of
form s. T h e m ost com m on form , w h ich is fou n d in approxim at ely 70% of t h e species exam in ed, is referred t o as t h e
Poly gon u m t ype (M ah esh w ari, 1950; Willem se an d van
Wen t , 1984; Reiser an d Fisch er, 1993). As sh ow n in Fig. 1,
t h e Poly gon u m -t ype fem ale gam et oph yt e is a seven -cell
1
T o w h om correspon den ce sh ou ld be addressed. Fax: (801) 5814668. E-m ail: drew s@bioscien ce.u t ah .edu .
136
st ru ct u re con sist in g of on e egg cell, t w o syn ergid cells,
t h ree an t ipodal cells, an d on e cen t ral cell.
T h e fem ale gam et oph yt e plays a cen t ral role in sexu al
reprodu ct ion in an giosperm s. D u rin g sexu al reprodu ct ion , t h e m ale gam et oph yt e is t ran sferred from t h e an t h er
t o t h e carpel’s st igm a, w h ere it form s a pollen t u be t h at
grow s t h rou gh t h e carpel t o deliver it s t w o sperm cells t o
t h e fem ale gam et oph yt e. Follow in g fert ilizat ion , t h e fem ale gam et oph yt e’s egg cell an d cen t ral cell give rise t o
t h e seed’s em bryo an d en dosperm , respect ively. T h e
fem ale gam et oph yt e plays a role in several st eps of t h is
reprodu ct ive processes in clu din g pollen t u be gu idan ce
(H u lsk am p et al., 1995; Ray et al., 1997), fert ilizat ion
(van Wen t an d Willem se, 1984; Ru ssell, 1993), t h e in du ct ion of seed developm en t (O h ad et al., 1996; C h au dh u ry
et al., 1997), an d gam et oph yt ic m at ern al con t rol (Ray,
1997; D rew s et al., 1998).
Fem ale gam et oph yt e developm en t con sist s of t w o ph ases
referred t o as m egasporogen esis an d m egagam et ogen esis.
0012-1606/ 98 $25.00
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137
Fem ale G am et oph y t e M u t an t s
FIG. 1. T h e A rab id opsis fem ale gam et oph yt e. (A,B) D epict ion s of a w ild-t ype fem ale gam et oph yt e at t h e seven -cell st age (st age FG 6).
D epict ed are cyt oplasm (ligh t gray areas), vacu oles (black areas), n u clei (dark gray areas), an d n u cleoli (w h it e areas w it h in t h e n u clei). T h e
fem ale gam et oph yt e in A is sh ow n in lon git u din al view ; it is orien t ed w it h it s adaxial su rface (adjacen t t o t h e fu n icu lu s; ad) t o t h e righ t ,
it s abaxial su rface (opposit e t h e fu n icu lu s; ab) t o t h e left . T h e fem ale gam et oph yt e in B is view ed from t h e abaxial direct ion . (C ) C LSM
im age of a w ild-t ype fem ale gam et oph yt e at t h e t erm in al st age (st age FG 7). T h e secon d syn ergid cell w as presen t bu t n ot in clu ded in t h is
im age. T h is im age con sist s of a sin gle 1.5-�m opt ical sect ion . All fem ale gam et oph yt es are orien t ed w it h t h eir ch alazal poles u p an d t h eir
m icropylar poles dow n . Abbreviat ion s: ab, abaxial su rface; ac, an t ipodal cells; ad, adaxial su rface; ch , ch alazal pole; cv, cen t ral cell vacu ole;
en , egg cell n u cleu s; ev, egg cell vacu ole; f, fu n icu lu s; m p, m icropylar pole; sen , secon dary en dosperm n u cleu s; sn , syn ergid cell n u cleu s;
sv, syn ergid cell vacu ole. Scale bar, 10 �m .
D u rin g m egasporogen esis, a diploid m egaspore m ot h er cell
u n dergoes m eiosis an d gives rise t o fou r h aploid m egaspores, t h ree of w h ich u n dergo cell deat h . D u rin g m egagam et ogen esis, t h e su rvivin g m egaspore u n dergoes t h ree
rou n ds of m it osis w it h ou t cyt ok in esis, produ cin g an eigh t n u cleat e cell. N u clear m igrat ion s, n u clear fu sion , an d cellu larizat ion t h en resu lt in t h e seven -cell st ru ct u re depict ed
in Fig. 1.
Lit t le is k n ow n abou t t h e h aploid-expressed gen es t h at
regu lat e an d m ediat e t h e m ajor even t s in m egagam et ogen esis (m it osis, polarit y est ablish m en t , n u clear m igrat ion , n u clear fu sion , cellu larizat ion , an d cell specifi cat ion ) an d fem ale gam et oph yt e fu n ct ion (pollen t u be
gu idan ce, fert ilizat ion , in du ct ion of seed developm en t ,
an d gam et oph yt ic m at ern al con t rol). A n u m ber of fem ale
gam et oph yt e m u t an t s h ave been iden t ifi ed in bot h A rab id opsis (Redei, 1965; C ast le et al., 1993; Kieber et al.,
1993; N iyogi et al., 1993; Sprin ger et al., 1995; M oore et
al., 1997) an d m aize (Sin glet on an d M an gelsdorf, 1940;
N elson an d C lary, 1952; Kerm icle, 1971), in dicat in g a
requ irem en t for h aploid-expressed gen es in m egagam et ogen esis an d fem ale gam et oph yt e fu n ct ion (D rew s et al.,
1998). H ow ever, a specifi c fu n ct ion is k n ow n on ly for t h e
PR L gen e, w h ich en codes a prot ein requ ired in D N A
replicat ion an d cell division (Sprin ger et al., 1995).
We h ave been u sin g A rab id opsis as a syst em t o iden t ify
an d st u dy h aploid-expressed gen es regu lat in g an d m edi-
at in g fem ale gam et oph yt e developm en t an d fu n ct ion .
Ext en sive descript ive st u dies of A rab id opsis fem ale gam et oph yt e st ru ct u re an d developm en t h ave been carried
ou t an d h ave sh ow n t h at A rab id opsis h as a Poly gon u m t ype fem ale gam et oph yt e (M isra, 1962; Poliak ova, 1964;
Webb an d G u n n in g, 1990; M an sfi eld an d Briart y, 1991;
M an sfi eld et al., 1991; M u rgia et al., 1993; Webb an d
G u n n in g, 1994; Sch n eit z et al., 1995; C h rist en sen et al.,
1997). Ph en ot ypic an alysis of fem ale gam et oph yt e m u t an t s in A rab id opsis is facilit at ed by t h e fact t h at an
A rab id opsis pist il con t ain s a large n u m ber of ovu les (50
t o 60) an d t h at t h e fem ale gam et oph yt es w it h in t h ese
ovu les develop syn ch ron ou sly (C h rist en sen et al., 1997).
Addit ion ally, con focal laser scan n in g m icroscopy procedu res h ave been developed t h at allow for t h e rapid
ch aract erizat ion of m u t an t ph en ot ypes, m ak in g largescale gen et ic screen s pract ical (C h rist en sen et al., 1997).
In t h is paper w e describe a procedu re t o iden t ify fem ale
gam et oph yt e m u t an t s an d report t h e isolat ion of fou r m u t an t s, fem ale gam et oph y t e1 (fem 1), fem 2, fem 3, an d fem 4.
We sh ow t h at all of t h e fem m u t at ion s exh ibit redu ced
t ran sm ission t h rou gh t h e fem ale gam et oph yt e an d t h at t h e
fem 3 an d fem 4 m u t at ion s also affect t h e m ale gam et oph yt e.
We also report t h e ph en ot ypic ch aract erizat ion of t h ese fou r
m u t an t s, as w ell as t h e gfa2, gfa3, gfa4, gfa5, an d gfa7
m u t an t s (Feldm an n et al., 1997).
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138
C h rist en sen , Su b ram an ian , an d D rew s
MATERIALS AN D METHOD S
Plant grow th and kanamycin selection. Plant growth conditions
and kanam ycin selection (50 �g/m l) were perform ed as previously
described (Christensen et al., 1997; Feldm ann et al., 1997).
Mut ant isolat ion. T h e fem 1 m u t an t w as isolat ed in a screen of
et h yl m et h an esu lfon at e (EM S)-m u t agen ized lin es. Lan dsberg
erect a seed w as soak ed in 0.1% EM S for 14 h , w ash ed for 3 h in
w at er, an d t h en sow n . O n each of 1000 M 1 plan t s, t h e M 2 seed
from a sin gle siliqu e w as collect ed. Five M 2 seeds from each M 1
siliqu e w ere plan t ed an d grow n u n t il at least 15 fl ow ers h ad been
self-pollin at ed. Wit h each M 2 plan t , at least t h ree siliqu es w ere cu t
open an d scored for t h e 50% desiccat ed ovu les ph en ot ype. T h e
fem 1 m u t an t w as back crossed fi ve gen erat ion s before gen et ic an d
m orph ological an alysis w as carried ou t .
T h e fem 2, fem 3, an d fem 4 m u t an t s w ere isolat ed in a screen of
T -D N A-m u t agen ized lin es as described u n der Resu lt s. T h e fem 2
an d fem 3 m u t an t s w ere isolat ed from lin es gen erat ed by Ken
Feldm an n an d obt ain ed from t h e A rab id opsis Biological Resou rce
C en t er in pools of 20. For t h ese lin es, w e screen ed an average of 53
plan t s in each of 57 pools (pools C S2361–C S2365, C S2461–C S2494,
C S2497–C S2501, C S2505, C S2507, C S2509 –C S2515, C S2520,
C S2522, C S2523, an d C S2534). We isolat ed fem 4 from a screen of
195 lin es gen erat ed an d provided by T om Jack .
G enet ic m ap p ing. We u sed sim ple sequ en ce len gt h polym orph ism s (Bell an d Eck er, 1994) an d cleaved am plifi ed polym orph ic
sequ en ces (Kon ieczn y an d Au su bel, 1993) t o m ap t h e m u t at ion s
w it h in t h e A rab id opsis gen om e. fem 1, in t h e Lan dsberg erect a
back grou n d, an d fem 2 an d fem 3, in t h e Wassilew sk ija (Ws) back grou n d, w ere crossed t o C olu m bia (C ol) ecot ype fem ales. fem 4 in
t h e C ol back grou n d w as crossed t o a Ws fem ale. For t h e fem 2, fem 3
an d fem 4 m u t an t s, on e Kan R F1 plan t from each cross w as select ed
an d again crossed t o a C ol fem ale (fem 2 an d fem 3) or a Ws fem ale
(fem 4). For t h e fem 1 m u t an t on e F1 plan t exh ibit in g t h e 50%
desiccat ed ovu les ph en ot ype w as select ed an d again crossed t o a
C ol fem ale. In all cases, t h e progen y resu lt in g from t h e secon d cross
con st it u t ed ou r m appin g popu lat ion .
D N A w as ext ract ed from eit h er Kan R plan t s (fem 2, fem 3, an d
fem 4) or plan t s exh ibit in g t h e 50% seed set ph en ot ype (fem 1) in t h e
m appin g popu lat ion s as described by M u rray an d T h om pson
(1980). T w en t y-m icrolit er polym erase ch ain react ion (PC R) react ion s w ere don e u sin g 1.6 �l of t em plat e (con cen t rat ion n ot
det erm in ed), 10 pm ol of each prim er (Research G en et ics, H u n t sville, AL), 4 �m ol each dN T P, 30 �m ol M gC l 2 , 0.5–1 u n it of Biolase
polym erase (ISC BioExpress, Kaysville, U T ), 1� Biolase react ion
bu ffer (ISC BioExpress), an d eit h er 1� t art razin e/ Ficoll or 1�
su crose/ creosol red (Idah o T ech n ologies, Idah o Falls, ID ). PC R
cyclin g param et ers w ere an in it ial den at u rat ion st ep at 94°C for 4
m in an d t h en 40 cycles of den at u rat ion at 94°C for 15 s, an n ealin g
at 55°C for 15 s, ext en sion at 72°C for 30 s, an d t h en a fi n al
ext en sion at 72°C for 4 m in on an M J Research , In c., PT C -100
Program m able T h erm al C on t roller.
At least 52 in dividu als from each m appin g popu lat ion w ere
t est ed again st each m ark er for lin k age. In all cases, t h e m ost closely
lin k ed m ark ers h ad recom bin at ion frequ en cies t h at w ere sign ifi can t ly differen t t h an 0.5 by �2 an alysis (P � 0.025). Recom bin at ion
frequ en cies w ere calcu lat ed an d con vert ed t o gen et ic m ap dist an ces
accordin g t o Koorn n eef an d St am (1992) an d Kosam bi (1944). fem 1
m apped bet w een n ga76 an d LFY3 on ch rom osom e 5. fem 1 an d
n ga76 h ad a recom bin at ion frequ en cy of 33.3 � 7.8 (m ap dist an ce �
40.2 cM , n � 54) an d fem 1 an d LFY3 h ad a recom bin at ion
frequ en cy of 25.0 � 6.0 (m ap dist an ce � 27.5 cM , n � 52). fem 2 an d
n ga8 on t h e t op of ch rom osom e 2 h ad a recom bin at ion frequ en cy of
9.6 � 4.1 (m ap dist an ce � 9.7 cM , n � 52). fem 3 an d n ga280 on
ch rom osom e 1 sh ow ed n o recom bin at ion (LO D score � 16.3, n �
54). Fin ally, fem 4 m apped bet w een n ga280 an d At h AT PASE on
ch rom osom e 1. fem 4 an d n ga280 h ad a recom bin at ion frequ en cy of
33.3 � 7.8 (m ap dist an ce � 40.2 cM , n � 54) an d fem 4 an d
At h AT PASE h ad a recom bin at ion frequ en cy of 24.1 � 5.8 (m ap
dist an ce � 26.3 cM , n � 54).
C onfocal laser scanning m icroscop y . T issu e preparat ion , m icroscopy, im age capt u re, an d fi gu re preparat ion w ere perform ed as
previou sly described (C h rist en sen et al., 1997) w it h t h e follow in g
m odifi cat ion s. For fem ale gam et oph yt e an alysis, carpel w alls w ere
separat ed from t h e sept u m prior t o fi xat ion by slicin g alon g bot h
sides of t h e pist il’s replu m w it h a 30-gau ge syrin ge n eedle. Aft er
clearin g, each placen t a w as dissect ed by rem ovin g t h e pedicel,
st yle, an d carpel w alls. For m ale gam et oph yt e an alysis, an t h ers
w ere fi xed ju st prior t o deh iscen ce an d w h ole m ou n t ed.
A ssignm ent of a fem ale gam et op hy t e st age t o a p ist il. We
previou sly sh ow ed t h at fem ale gam et oph yt e developm en t w it h in a
pist il is fairly syn ch ron ou s (C h rist en sen et al., 1997). In m ost cases,
a sin gle fem ale gam et oph yt e st age predom in at es (�75% ) w it h in a
pist il. U n der t h is circu m st an ce, w e assign on e fem ale gam et oph yt e
st age t o a pist il. In som e cases, a sin gle st age does n ot predom in at e
becau se t h e pist il is at a t ran sit ion bet w een t w o st ages. U n der t h is
circu m st an ce, w e assign t h e pist il t h e t w o st ages t h at t oget h er
con st it u t e �75% of t h e fem ale gam et oph yt es.
Recip rocal crosses. T o preven t t h e possibilit y of in advert en t
self-pollin at ion or cross-con t am in at ion , sin gle fem ale paren t s w ere
isolat ed in 2-lit er soda bot t les, all fl ow ers on t h e prim ary in fl orescen ce w ere em ascu lat ed daily before deh iscen ce an d pollin at ed t h e
follow in g day, an d secon dary in fl orescen ces w ere rem oved. In
crosses bet w een m u t an t m ales an d w ild-t ype fem ales, t h e fem ale
paren t w as m ale st erile1-1 (van der Veen an d Wirt z, 1968).
RESU LTS
Isolat ion of Fem ale G am et op hy t e Mut ant s
We screen ed for lin es con t ain in g fem ale gam et oph yt e
let h al m u t at ion s. O u r screen con sist ed of t w o st eps. As a
prim ary screen , w e open ed siliqu es an d screen ed for plan t s
w it h redu ced seed set . O n a plan t h et erozygou s for a fem ale
gam et oph yt e m u t at ion , abou t 50% of it s fem ale gam et oph yt es are w ild t ype an d fu n ct ion al, an d approxim at ely
50% are m u t an t an d n on fu n ct ion al. Follow in g fert ilizat ion ,
fu n ct ion al ovu les develop in t o seeds. By con t rast , ovu les
h arborin g defect ive fem ale gam et oph yt es fail t o u n dergo
seed developm en t an d desiccat e in st ead. T h u s, siliqu es
from h et erozygou s fem ale gam et oph yt e m u t an t s con t ain
approxim at ely 50% n orm al seeds an d 50% desiccat ed
ovu les.
T h e secon dary screen w as based on t h e con cept t h at
fem ale gam et oph yt e m u t an t s exh ibit alt ered segregat ion
pat t ern s. For exam ple, self-pollin at ion of a h et erozygou s
fem ale gam et oph yt e m u t an t produ ces h et erozygou s an d
w ild-t ype progen y in a 1:1 rat io (if t h e m u t at ion is fu lly
pen et ran t an d does n ot affect t h e m ale gam et oph yt e). T h e
lin es w e screen ed w ere m u t agen ized w it h a T -D N A con t ain in g t h e ch im eric n pt II gen e w h ich con fers k an am ycin
resist an ce (Feldm an n et al., 1997). T h u s, self-pollin at ion of
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139
Fem ale G am et oph y t e M u t an t s
TABLE 1
M u t an t s D iscu ssed in T h is Paper
M u t an t
fem ale gam et oph y t e1 (fem 1)
fem ale gam et oph y t e2 (fem 2)
fem ale gam et oph y t e3 (fem 3)
fem ale gam et oph y t e4 (fem 4)
gam et oph y t ic fact or2 (gfa2)
gam et oph y t ic fact or3 (gfa3)
gam et oph y t ic fact or4 (gfa4)
gam et oph y t ic fact or5 (gfa5)
gam et oph y t ic fact or7 (gfa7)
a
Isolat e
n o.
M u t agen
M u t an t
class a
864
2462-5
2465-35
T J160
102
13
84
114
21
EM S
T -D N A
T -D N A
T -D N A
T -D N A
T -D N A
T -D N A
T -D N A
T -D N A
FG S
FG S
GG
GG
GG
GG
GG
GG
GG
Sou rce
T h is paper
T h is paper
T h is paper
T h is paper
(Feldm an n
(Feldm an n
(Feldm an n
(Feldm an n
(Feldm an n
et
et
et
et
et
al.,
al.,
al.,
al.,
al.,
1997)
1997)
1997)
1997)
1997)
FG S, fem ale gam et oph yt e-specifi c; G G , gen eral gam et oph yt ic.
a h et erozygou s fem ale gam et oph yt e m u t an t produ ces k an am ycin resist an t (Kan R) an d k an am ycin sen sit ive (Kan S)
progen y in a 1:1 rat io (if t h e m u t at ion is fu lly pen et ran t an d
does n ot affect t h e m ale gam et oph yt e). By con t rast , selfpollin at ion of a h et erozygou s sporoph yt ic m u t an t (e.g., a
m u t an t defect ive in fl ow er developm en t ) produ ces Kan R
an d Kan S progen y in a 3:1 rat io.
All pot en t ial m u t an t s iden t ifi ed in t h e prim ary screen
w ere allow ed t o self-pollin at e, 100 –200 progen y seeds w ere
germ in at ed in t h e presen ce of Kan , an d Kan resist an ce
rat ios (Kan R/ Kan S) w ere scored. Alt h ou gh w e w ere prim arily in t erest ed in lin es in w h ich Kan R/ Kan S w as 1.0, w e also
exam in ed lin es in w h ich Kan R/ Kan S w as sligh t ly h igh er or
low er t h an 1.0 becau se, as discu ssed below , fem ale gam et oph yt e m u t at ion s can be part ially pen et ran t an d can also
affect t h e m ale gam et oph yt e.
T h ree lin es, fem 2, fem 3, an d fem 4, w ere iden t ifi ed t h at
exh ibit ed redu ced seed set an d in w h ich Kan R/ Kan S w as
less t h an 1.5. We also iden t ifi ed on e fem ale gam et oph yt e
m u t an t (fem 1) in an EM S screen (see M at erials an d M et h ods) an d obt ain ed fi ve addit ion al fem ale gam et oph yt e m u t an t s (gfa2, gfa3, gfa4, gfa5, an d gfa7) from ot h er sou rces, as
list ed in T able 1.
We det erm in ed t h e gen et ic m ap posit ion s of t h e fem
m u t at ion s. All fou r m u t at ion s m apped t o locat ion s in t h e
gen om e t h at w ere differen t from each ot h er an d from t h e
gfa1– gfa7 m u t at ion s (Feldm an n et al., 1997), in dicat in g
t h at t h ey are n ot allelic (see M at erials an d M et h ods). O f t h e
ot h er m u t an t s discu ssed in t h is paper, on ly gfa3 an d gfa7
m ap t o sim ilar posit ion s (Feldm an n et al., 1997).
Penet rance in t he Fem ale G am et op hy t e
T o det erm in e t h e pen et ran ce of t h e fem m u t at ion s in t h e
fem ale gam et oph yt e, w e crossed h et erozygou s m u t an t
plan t s as fem ale paren t s w it h w ild-t ype m ales an d scored
t h e n u m ber of F1 progen y t h at w ere h et erozygou s (Kan R or
exh ibit ed 50% seed set ) an d h om ozygou s w ild t ype (Kan S or
exh ibit ed 100% seed set ). T h e dat a from t h ese crosses are
su m m arized in T able 2. Wit h all m u t an t s t est ed, t h e rat io of
h et erozygou s t o h om ozygou s w ild-t ype (Kan R/ Kan S) progen y w as �1.0, in dicat in g t h at t h ese m u t at ion s exh ibit ed
redu ced t ran sm ission t h rou gh t h e fem ale gam et oph yt e an d
t h u s affect ed t h e fem ale gam et oph yt e. Wit h t h e fem 1 an d
fem 4 m u t an t s, n o t ran sm ission of t h e m u t at ion w as observed, in dicat in g t h at t h e fem 1 an d fem 4 m u t at ion s w ere
100% pen et ran t in t h e fem ale gam et oph yt e. By con t rast ,
Link age A naly sis
T o det erm in e w h et h er t h e fem 2–fem 4 m u t at ion s w ere
lin k ed t o t h e n pt II gen e, w e crossed h et erozygou s fem ale
gam et oph yt e m u t an t s w it h w ild t ype, germ in at ed t h e F1
progen y in t h e presen ce of Kan , t ran sferred 100 Kan R F1
progen y t o dirt , an d scored t h ese plan t s for redu ced seed set .
Wit h t h e fem 2–fem 4 m u t an t s, all 100 of t h e F1 progen y
scored exh ibit ed redu ced seed set , in dicat in g t h at in each of
t h ese lin es, t h e n pt II gen e w as lin k ed t o t h e fem ale gam et oph yt e m u t at ion w it h in 3.7 cM at t h e 95% con fi den ce level.
T h e t igh t lin k age bet w een t h e n pt II gen e an d t h e fem ale
gam et oph yt e m u t at ion s allow ed u s, in su bsequ en t experim en t s, t o u se t h e Kan R t rait as a m ark er for t h e presen ce of
t h e m u t at ion s.
TABLE 2
Resu lt s from C rosses of H et erozygou s Fem ale G am et oph yt e
M u t an t s as Fem ale Paren t s w it h Wild-T ype M ales
N o. of progen y
M u t an t
H et erozygou s
H om ozygou s
H et / H om o a
Pen et ran ce
in FG
fem 1
fem 2
fem 3
fem 4
0
273
225
0
206
486
388
111
0
0.56
0.58
0
100
44
42
100
a
Rat io of h et erozygou s t o h om ozygou s progen y.
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140
C h rist en sen , Su b ram an ian , an d D rew s
TABLE 3
Resu lt s from C rosses of H et erozygou s Fem ale G am et oph yt e
M u t an t s as M ale Paren t s w it h Wild-T ype Fem ales
N o. of progen y
M u t an t
H et erozygou s
H om ozygou s
H et / H om o a
Pen et ran ce
in M G
fem 1
fem 2
fem 3
fem 4
219
342
340
238
221
303
606
412
0.99
1.13
0.56
0.58
1b
0
44
42
a
b
Rat io of h et erozygou s t o h om ozygou s progen y.
N ot sign ifi can t ly differen t t h an 0% .
w it h t h e fem 2 an d fem 3 m u t an t s, at least 56% t ran sm ission
of t h e m u t at ion t h rou gh t h e fem ale gam et oph yt e w as
observed, in dicat in g t h at t h e correspon din g m u t at ion s w ere
part ially pen et ran t in t h e fem ale gam et oph yt e. We, in
addit ion , carried ou t crosses w it h t h e gfa2, gfa3, gfa4, gfa5,
an d gfa7 m u t an t s an d obt ain ed pen et ran ce valu es sim ilar t o
t h ose report ed previou sly (Feldm an n et al., 1997).
Fem ale G am et op hy t e Sp ecificit y
T o det erm in e w h et h er t h e fem m u t at ion s also affect t h e
m ale gam et oph yt e, w e crossed h et erozygou s m u t an t plan t s
as m ale paren t s w it h w ild-t ype fem ales an d scored t h e
n u m ber of F1 progen y t h at w ere h et erozygou s (Kan R or
exh ibit ed 50% seed set ) an d h om ozygou s w ild t ype (Kan S or
exh ibit ed 100% seed set ). T h e dat a from t h ese crosses are
su m m arized in T able 3. Wit h t h e fem 1 an d fem 2 m u t an t s,
h et erozygou s an d h om ozygou s w ild-t ype progen y w ere
presen t in approxim at ely equ al proport ion s in t h e F1 gen erat ion , in dicat in g t h at t h e fem 1 an d fem 2 m u t at ion s do
n ot affect t h e m ale gam et oph yt e. By con t rast , w it h t h e
fem 3 an d fem 4 m u t an t s, t h e rat io of h et erozygou s t o
h om ozygou s w ild-t ype (Kan R/ Kan S) progen y w as �0.6,
in dicat in g t h at t h e correspon din g m u t at ion s exh ibit ed redu ced t ran sm ission t h rou gh t h e m ale gam et oph yt e an d
t h u s affect ed t h e m ale gam et oph yt e.
In su m m ary, w e h ave iden t ifi ed t w o classes of fem ale
gam et oph yt e m u t at ion s. O n e class affect s t h e fem ale gam et oph yt e bu t n ot t h e m ale gam et oph yt e an d t h e secon d
class affect s bot h gam et oph yt es. We refer t o t h ese t w o
classes as fem ale gam et oph yt e-specifi c m u t at ion s an d gen eral gam et oph yt ic m u t at ion s, respect ively. T h e class t h at
each of t h e fem ale gam et oph yt e m u t at ion s falls w it h in is
list ed in T able 1.
Megagam et ogenesis in t he fem and gfa Mut ant s
Fem ale gam et oph yt e m u t at ion s cou ld affect t h e fem ale
gam et oph yt e eit h er by disru pt in g m egagam et ogen esis or by
in t erferin g w it h on e of t h e fem ale gam et oph yt e’s fu n ct ion s.
T o dist in gu ish bet w een t h ese possibilit ies, w e an alyzed t h e
t erm in al ph en ot ypes of t h e fem ale gam et oph yt e m u t an t s.
T o do t h is, w e allow ed t h e fem ale gam et oph yt es w it h in
h et erozygou s pist ils t o progress t o t h e t erm in al developm en t al st age (st age FG 7, Fig. 2A) an d exam in ed at least 80
fem ale gam et oph yt es for each m u t an t . T h e fem 1, fem 2,
fem 3, fem 4, gfa2, gfa3, gfa4, gfa5, an d gfa7 m u t an t s h ad
m orph ologically abn orm al fem ale gam et oph yt es at t h e t erm in al developm en t al st age, in dicat in g t h at t h e m u t at ion s
in t h ese lin es affect m egagam et ogen esis. T h e t erm in al
ph en ot ypes of t h e fem ale gam et oph yt e m u t an t s are su m m arized in T able 4 an d are discu ssed in det ail below .
T o det erm in e t h e developm en t al st age at w h ich m egagam et ogen esis depart s from w ild t ype in t h e fem an d gfa
m u t an t s, w e an alyzed m egagam et ogen esis at all developm en t al st ages in h et erozygou s pist ils. We previou sly
sh ow ed t h at fem ale gam et oph yt e developm en t w it h in a
pist il is syn ch ron ou s (C h rist en sen et al., 1997), w h ich
allow ed u s t o u se w ild-t ype fem ale gam et oph yt es t o det erm in e t h e developm en t al st age of m u t an t fem ale gam et oph yt es w it h in t h e sam e pist il. Each developm en t al st age
w as an alyzed by con focal laser scan n in g m icroscopy
(C LSM ). U sin g t h is procedu re, n u cleoli appear w h it e, cyt oplasm appears gray, an d vacu oles appear black (Figs. 1C , 3,
an d 4) (C h rist en sen et al., 1997). T h e resu lt s of t h is an alysis
are su m m arized in T able 5. C LSM im ages of m u t an t fem ale
gam et oph yt es are sh ow n in Figs. 3 an d 4.
Megagam et ogenesis in w ild t y p e. M egagam et ogen esis
in w ild-t ype A rab id opsis h as been described (M isra, 1962;
Poliak ova, 1964; Webb an d G u n n in g, 1990; M an sfi eld et al.,
1991; M u rgia et al., 1993; Webb an d G u n n in g, 1994;
Sch n eit z et al., 1995; C h rist en sen et al., 1997) an d h as been
divided in t o seven m orph ologically dist in ct st ages t h at are
depict ed in Fig. 2A (C h rist en sen et al., 1997). First , t h e
su rvivin g m egaspore (st age FG 1) u n dergoes m it osis t o produ ce a t w o-n u cleat e cell (st age FG 2). Sh ort ly t h ereaft er, t h e
t w o n u clei separat e t o t h e ch alazal an d m icropylar poles
an d a vacu ole form s in t h e cen t er (st age FG 3). T h e developin g fem ale gam et oph yt e t h en u n dergoes secon d (st age FG 4)
an d t h ird rou n ds of m it osis. T h is resu lt s in an eigh t n u cleat e cell w it h fou r n u clei at each of t w o poles separat ed
by a large cen t ral vacu ole (begin n in g of st age FG 5). N ext ,
on e n u cleu s from each pole (t h e polar n u clei) m igrat es
t ow ard t h e fem ale gam et oph yt e’s cen t er an d t h e em bryo sac
becom es cellu larized. T h e polar n u clei even t u ally fu se an d
t h e fem ale gam et oph yt e con sist s of seven cells an d seven
n u clei (st age FG 6; Figs. 1A an d 1B). As a fi n al developm en t al st ep, t h e t h ree an t ipodal cells u n dergo cell deat h . T h u s,
t h e m at u re A rab id opsis fem ale gam et oph yt e (st age FG 7)
con sist s of on e h aploid egg cell, t w o h aploid syn ergid cells,
an d on e diploid cen t ral cell (Figs. 1C an d 2B). M egagam et ogen esis in A rab id opsis occu rs over a period of approxim at ely 3.5 days (C . A. C h rist en sen an d G . N . D rew s,
u n pu blish ed resu lt s).
T o det erm in e t h e ext en t t o w h ich abn orm al fem ale
gam et oph yt e developm en t occu rs in w ild t ype, w e an alyzed
�1000 w ild-t ype fem ale gam et oph yt es en com passin g all
developm en t al st ages. U n der ou r grow t h con dit ion s (C h ris-
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141
Fem ale G am et oph y t e M u t an t s
FIG. 2. D epict ion s of m egagam et ogen esis in w ild-t ype (A,B) an d fem ale gam et oph yt e m u t an t ph en ot ypes at t h e t erm in al st age (C –F). (A)
M egagam et ogen esis st ages (C h rist en sen et al., 1997). (B) Lon git u din al view of a w ild-t ype fem ale gam et oph yt e at t h e t erm in al st age (st age
FG 7). (C ) T erm in al ph en ot ype of fem 2, fem 3, gfa4, an d gfa5 fem ale gam et oph yt es. (D ) T erm in al ph en ot ype of gfa2, gfa3, an d gfa7 fem ale
gam et oph yt es. (E) T erm in al ph en ot ype of gfa3 an d gfa7 fem ale gam et oph yt es. (F) T erm in al ph en ot ype of t h e fem 4 m u t an t . D epict ed are
cyt oplasm (ligh t gray areas), vacu oles (black areas), n u clei (dark gray areas), an d n u cleoli (w h it e areas w it h in t h e n u clei). All fem ale
gam et oph yt es are orien t ed w it h t h eir ch alazal poles u p an d t h eir m icropylar poles dow n . Abbreviat ion s: ac, an t ipodal cells; cc, cen t ral cell;
ce, cell in sim ilar posit ion an d polarit y t o egg cell; cs, cell in sim ilar posit ion an d polarit y t o syn ergid cell; ec, egg cell; pn , polar n u cleu s;
sc, syn ergid cell; sen , secon dary en dosperm n u cleu s.
t en sen et al., 1997), less t h an 1% of t h e ovu les w e observed
con t ain ed a fem ale gam et oph t ye t h at h ad u n dergon e abn orm al developm en t . O f t h ose t h at developed abn orm ally, all
abort ed at t h e on e-n u cleat e st age an d n on e h ad a defect in a
lat er developm en t al st ep (i.e., polar n u clei m igrat ion , cellu larizat ion , polar n u clei fu sion , or an t ipodal cell deat h ).
gfa2. At t h e t erm in al developm en t al st age (st age FG 7)
approxim at ely h alf of t h e em bryo sacs in gfa2/ G FA 2 pist ils
w ere ph en ot ypically m u t an t (T able 5), con sist en t w it h t h e
100% pen et ran ce of t h e gfa2 m u t at ion in t h e fem ale gam et oph yt e (Feldm an n et al., 1997). T h e t erm in al ph en ot ype
of gfa2 fem ale gam et oph yt es is sh ow n in Fig. 3B an d
depict ed in Fig. 2D . In m at u re gfa2 fem ale gam et oph yt es,
t h e overall m orph ology of t h e egg cell, cen t ral cell, an d
syn ergid cells w as n orm al (Fig. 3B an d dat a n ot sh ow n ).
H ow ever, in con t rast t o w ild-t ype fem ale gam et oph yt es at
st age FG 7, t h e n u cleoli of t h e t w o polar n u clei w ere u n fu sed
(Fig. 3B). Becau se Fig. 3B is a project ion of several C LSM
opt ical sect ion s, t h e n u clear m em bran e bou n dary w as n ot
dist in gu ish able. Figu res 3C an d 3D are sin gle opt ical sect ion s of m at u re gfa2 fem ale gam et oph yt es sh ow in g t h at t h e
t w o polar n u clei, as w ell as t h e n u cleoli, w ere separat e.
T h u s, t h e gfa2 m u t at ion appears t o specifi cally affect fu sion
of t h e polar n u clei.
T o det erm in e t h e st age in w h ich fem ale gam et oph yt e
developm en t depart s from w ild t ype, w e an alyzed gfa2/
G FA 2 pist ils con t ain in g fem ale gam et oph yt es bet w een
st ages FG 1 an d FG 7. In gfa2/ G FA 2 pist ils con t ain in g fem ale
gam et oph yt es bet w een st ages FG 6 an d FG 7 (pist il st ages
FG 6/ 7 an d FG 7), approxim at ely h alf (109/ 207) of t h e fem ale
gam et oph yt es observed w ere abn orm al (T able 5). By con t rast , in gfa2/ G FA 2 pist ils con t ain in g fem ale gam et oph yt es
at st age FG 5 an d earlier (pist il st ages FG 1-FG 5/ 6), essen t ially all (283/ 291) fem ale gam et oph yt es observed w ere
m orph ologically w ild t ype (T able 5). We t h erefore con clu de
t h at gfa2 fem ale gam et oph yt es begin t o develop abn orm ally
begin n in g at t h e t ran sit ion from st age FG 5 t o st age FG 6,
w h ich is t h e t im e w h en t h e polar n u clei fu se (Fig. 2A).
In ou r an alysis of developin g fem ale gam et oph yt es, t h e
m ajor defect observed w as a failu re of t h e polar n u clei t o
fu se. In w ild-t ype fem ale gam et oph yt es, an t ipodal cell degen erat ion occu rs su bsequ en t t o polar n u clei fu sion . In gfa2
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142
C h rist en sen , Su b ram an ian , an d D rew s
FIG. 3. C LSM im ages of fem 3, gfa2, an d gfa3 fem ale gam et oph yt es. (A) T erm in al ph en ot ype of a fem 3 fem ale gam et oph yt e. T h is im age
is a project ion of t w o 1.5-�m opt ical sect ion s. (B) T erm in al ph en ot ype of a gfa2 fem ale gam et oph yt e. T h is im age is a project ion of fou r
1.5-�m opt ical sect ion s. (C ,D ) Sin gle opt ical sect ion s sh ow in g a m agn ifi ed view of t h e u n fu sed polar n u clei in t w o gfa2 fem ale
gam et oph yt es. T h e im age in C is a sin gle opt ical sect ion of t h e project ion sh ow n in B. Arrow h eads sh ow t h e posit ion of t h e n u clear
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143
Fem ale G am et oph y t e M u t an t s
TABLE 4
T erm in al Ph en ot ypes of Fem ale G am et oph yt e M u t an t s
M u t an t
Ph en ot ype
fem 1
fem 2
fem 3
fem 4
Fem ale gam et oph yt e absen t . Em bryo sac cavit y fi lled w it h h igh ly au t ofl u orescen t m at erial.
O n e-n u cleat e fem ale gam et oph yt e.
O n e-n u cleat e fem ale gam et oph yt e.
At t h e m icropylar pole, cell m orph ology, in clu din g n u clear posit ion in g, vacu olar an d cellu lar sh ape, an d vacu ole
n u m ber w as irregu lar. N u clear an d cell n u m bers correspon d t o t h ose of st ages FG 5, FG 6, or FG 7.
Polar n u clei fail t o fu se.
In m ost (59% ), t h e polar n u clei fail t o fu se. In som e (22% ), t h e em bryo sac h as fi ve n u clei in approxim at ely t h e correct
posit ion s (t o be t h e egg, syn ergid an d u n fu sed polar n u clei), bu t appears t o be u n cellu larized. In som e (19% ), t h e
ovu les lack ed fem ale gam et oph yt es.
O n e-n u cleat e fem ale gam et oph yt e.
O n e-n u cleat e fem ale gam et oph yt e.
In m ost (58% ), t h e polar n u clei fail t o fu se. In som e (28% ), t h e em bryo sac h as fi ve n u clei in approxim at ely t h e correct
posit ion s (t o be t h e egg, syn ergid an d u n fu sed polar n u clei), bu t appears t o be u n cellu larized. In som e (14% ), t h e
ovu les lack ed fem ale gam et oph yt es.
gfa2
gfa3
gfa4
gfa5
gfa7
fem ale gam et oph yt es, an t ipodal cell degen erat ion also occu rs, alt h ou gh it appeared t o occu r m ore slow ly t h an in
w ild t ype (dat a n ot sh ow n ).
gfa3. U n der ou r grow t h con dit ion s, t h e pen et ran ce of
t h e gfa3 m u t at ion in t h e fem ale gam et oph yt e w as 82% ;
t h u s, t h e expect ed frequ en cy of ph en ot ypically m u t an t
em bryo sacs in gfa3/ G FA 3 pist ils w as 41% . H ow ever, at t h e
t erm in al developm en t al st age (st age FG 7), approxim at ely
30% of t h e fem ale gam et oph yt es in gfa3/ G FA 3 pist ils w ere
ph en ot ypically m u t an t (T able 5), su ggest in g t h at som e
n on fu n ct ion al gfa3 fem ale gam et oph yt es w ere m orph ologically n orm al at t h e resolu t ion of C LSM .
T h e ph en ot ypes exh ibit ed by gfa3 fem ale gam et oph yt es
at t h e t erm in al developm en t al st age are sh ow n in Figs.
3E–3H an d depict ed in Figs. 2D an d 2E. T h e predom in an t
(54/ 92) defect observed w as failu re of t h e polar n u clei t o
fu se (Figs. 2D an d 3E). As w it h gfa2 m u t an t s, in dividu al
opt ical sect ion s sh ow ed t h at t h e polar n u clei w ere adjacen t
t o on e an ot h er bu t rem ain ed separat e (Fig. 3F). T w o less
frequ en t t erm in al ph en ot ypes w ere also observed at t h e
t erm in al st age (T able 4). T h e fi rst w as fem ale gam et oph yt es
t h at w ere u n cellu larized bu t con t ain ed fi ve n u clei t h at w ere
in approxim at ely t h e correct posit ion s t o be t h e egg
n u cleu s, syn ergid n u clei, an d u n fu sed polar n u clei (Figs. 2E
an d 3G ). T h e secon d w as an ovu le lack in g a fem ale gam et oph yt e (Fig. 3H ).
T h e defect s exh ibit ed by developin g gfa3 fem ale gam et oph yt es are su m m arized in T able 6. T h e m ajor defect observed occu rred du rin g st age FG 6. D u rin g st age FG 6 in w ild
t ype, t h e polar n u clei are fu sed an d t h e an t ipodal cells are
u n dergoin g cell deat h . By st age FG 7, t h e an t ipodal cells are
com plet ely degen erat ed (Fig. 2A). In gfa3/ G FA 3 pist ils at
st age FG 7, 304 fem ale gam et oph yt es w ere observed t h at
lack ed an t ipodal cells, an d of t h ese 54 h ad u n fu sed polar
n u clei (“ u n fu sed polar n u clei” colu m n in T able 6, Fig. 3E).
Prior t o st age FG 6, t w o less frequ en t defect s w ere observed.
T h e fi rst w as com pressed em bryo sacs at t h e fou r-n u cleat e
st age (“ Fou r n u clei an d com pressed” colu m n in T able 6)
an d t h e secon d w as u n cellu larized fem ale gam et oph yt es at
lat e st age FG 5 (“ U n cellu larized” colu m n in T able 6, Fig. 3I).
Based on frequ en cy an d developm en t al progression , t h ese
t w o defect s probably gave rise t o t h e ovu les lack in g fem ale
gam et oph yt es (“ N o FG ” colu m n in T able 6, Fig. 3H ) an d t h e
u n cellu larized fem ale gam et oph yt es observed at t h e t erm in al st age (“ U n cellu larized” colu m n in T able 6, Fig. 3G ),
respect ively.
gfa7. T h e gfa7 m u t an t exh ibit ed t h e sam e t h ree t erm in al ph en ot ypes t h at w ere exh ibit ed by t h e gfa3 m u t an t in
bou n daries. (E) T erm in al ph en ot ype of a gfa3 fem ale gam et oph yt e. T h is im age is a project ion of fi ve 1.5-�m opt ical sect ion s. (F) Sin gle
opt ical sect ion sh ow in g a m agn ifi ed view of t h e u n fu sed polar n u clei in a gfa3 fem ale gam et oph yt e. Arrow h eads sh ow t h e posit ion of t h e
n u clear bou n daries. (G ) T erm in al ph en ot ype of a gfa3 fem ale gam et oph yt e. T h is im age is a project ion of eigh t 1.5-�m opt ical sect ion s. (H )
T erm in al ph en ot ype of a gfa3 fem ale gam et oph yt e. T h is im age con sist s of a sin gle opt ical sect ion . (I) Ph en ot ype of a gfa3 fem ale
gam et oph yt e at pist il st age FG 5. T h is im age is a project ion of eigh t 1.5-�m opt ical sect ion s. All fem ale gam et oph yt es are orien t ed w it h t h eir
ch alazal poles u p an d m icropylar poles dow n an d t h eir abaxial su rface t o t h e left an d adaxial su rface t o t h e righ t . Abbreviat ion s: en , egg cell
n u cleu s; cv, cen t ral cell vacu ole; m fg, m u t an t fem ale gam et oph yt e; n , n u cleu s; n e, n u cleu s in sim ilar posit ion t o egg n u cleu s; n pn , n u cleu s
in sim ilar posit ion t o polar n u cleu s; n s, n u cleu s in sim ilar posit ion t o syn ergid n u cleu s; pn , polar n u cleu s; sn , syn ergid cell n u cleu s; v,
vacu ole. Scale bars, 10 �m .
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144
C h rist en sen , Su b ram an ian , an d D rew s
FIG. 4. C LSM im ages of fem 1 an d fem 4 fem ale gam et oph yt es. (A) T erm in al ph en ot ype of a fem 1 fem ale gam et oph yt e. T h is im age is a
project ion of six 1.5-�m opt ical sect ion s. (B,C ) Ph en ot ypes of t w o fem 1 fem ale gam et oph yt es at pist il st age FG 5/ 6. Each im age is a
project ion of fou r 1.5 �m opt ical sect ion s. (D –H ) Ph en ot ypes of fem 4 fem ale gam et oph yt es at t h e t erm in al st age. D u e t o abn orm al cellu lar
m orph ologies, dist in ct cellu lar iden t it ies cou ld n ot be assign ed. T h e posit ion an d overall polarit y of cell e are sim ilar t o t h ose of t h e egg cell.
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145
Fem ale G am et oph y t e M u t an t s
TABLE 5
Su m m ary of C LSM An alysis of Fem ale G am et oph yt e M u t an t s
M u t an t
Pist il st age a
N o. of
pist ils
an alyzed
fem 1
FG 3–FG 4/ 5
FG 5
FG 5/ 6
FG 6/ 7
FG 7
FG 1–FG 3/ 4
FG 4
FG 4/ 5
FG 5
FG 5/ 6
FG 6
FG 6/ 7
FG 7
FG 1–FG 4/ 5
FG 5
FG 5/ 6
FG 6/ 7
FG 7
FG 1–FG 4
FG 4/ 5
FG 5
FG 5/ 6
FG 7
9
4
4
9
5
11
4
4
5
2
2
4
10
15
6
2
3
11
7
4
5
7
18
fem 4
gfa2
gfa3
a
N o. of
n orm al FG s
observed
N o. of
abn orm al FG s
observed
% Abn orm al
108
60
41
74
38
158
51
37
45
19
11
18
86
197
60
26
21
77
83
77
44
66
212
2
0
33
77
42
1
4
7
13
13
10
17
86
3
1
5
19
90
0
3
10
12
92
2
0
45
51
53
�1
7
16
22
41
48
49
50
2
2
16
48
54
0
4
19
15
30
Assign m en t of a pist il st age is described u n der M at erials an d M et h ods.
approxim at ely t h e sam e frequ en cies (T able 4). Sin ce t h e
gfa3 an d gfa7 m u t an t s h ave an iden t ical t erm in al ph en ot ype (T able 4) an d also h ave t h e sam e m ap posit ion (Feldm an n et al., 1997), t h ey m ost lik ely h arbor m u t at ion s in t h e
sam e gen e. For t h is reason , w e exam in ed earlier developm en t al st ages on ly in t h e gfa3 m u t an t .
fem 1. At t h e t erm in al developm en t al st age (st age FG 7),
approxim at ely h alf of t h e fem ale gam et oph yt es in fem 1/
FEM 1 pist ils w ere ph en ot ypically m u t an t (T able 5), con sist en t w it h t h e 100% pen et ran ce of t h e fem 1 m u t at ion . As
sh ow n in Fig. 4A, fem 1 fem ale gam et oph yt es at t h e t erm in al st age w ere com plet ely degen erat ed an d t h eir em bryo sac
cavit ies w ere fi lled w it h au t ofl u orescen t m at erial.
As sh ow n in T able 5, fem 1 fem ale gam et oph yt es begin t o
develop abn orm ally at approxim at ely lat e st age FG 5 or
st age FG 6, w h ich is abou t t h e t im e w h en t h e polar n u clei
fu se (Fig. 2A). T h e m ost com m on ly observed (27/ 33) ph en ot ype at t h is developm en t al t im e is sh ow n in Fig. 4B. As
w it h lat er st ages (Fig. 4A), t h e fem ale gam et oph yt e w as
com plet ely degen erat ed an d t h e em bryo sac cavit y w as
T h e posit ion s an d overall polarit ies of cells s1 an d s2 are sim ilar t o t h ose of t h e syn ergids. (D –F) Serial opt ical sect ion s of t h e m icropylar
h alf of on e fem 4 fem ale gam et oph yt e. (D ) A sin gle opt ical sect ion sh ow in g cell s1 (n s1 an d vs1). (E) A project ion of t w o 1.5-�m opt ical
sect ion s sh ow in g cell e (n e an d ve). (F) A project ion of t w o 1.5-�m opt ical sect ion s sh ow in g cell s2 (n s2 an d vs2). T h e vacu ole labeled v w as
in addit ion t o t h e t h ree vacu oles n orm ally seen in t h e egg apparat u s. T h e t w o vacu oles labeled cv w ere con n ect ed (n ot sh ow n ). (G ) In t h is
fem 4 fem ale gam et oph yt e, t h e polar n u clei h ave fu sed (sen ) an d t h e an t ipodal cells h ave degen erat ed. T h is im age is a project ion of t h ree
1.5-�m opt ical sect ion s. An addit ion al syn ergid cell w as presen t bu t n ot in clu ded in t h e project ion . (H ) In t h is fem 4 fem ale gam et oph yt e,
t h e polar n u clei (n pn ) w ere u n fu sed an d t h e an t ipodal cells (an ) w ere presen t . T h is im age is a project ion of fi ve 1.5-�m opt ical sect ion s. All
fem ale gam et oph yt es are orien t ed w it h t h eir ch alazal poles u p an d m icropylar poles dow n an d t h eir abaxial su rface t o t h e left an d adaxial
su rface t o t h e righ t . Abbreviat ion s: an , an t ipodal cell n u cleu s; cv, cen t ral cell vacu ole; m fg, m u t an t fem ale gam et oph yt e; n e, n u cleu s in
sim ilar posit ion t o egg n u cleu s; n pn , n u cleu s in sim ilar posit ion t o polar n u cleu s; n s, n u cleu s in sim ilar posit ion t o syn ergid n u cleu s; n s1,
n u cleu s of cell s1; n s2, n u cleu s of cell s2; sen , secon dary en dosperm n u cleu s; v, vacu ole; ve, vacu ole in sim ilar posit ion t o egg cell vacu ole;
vs, vacu ole in sim ilar posit ion t o syn ergid cell vacu ole; vs1, vacu ole of cell s1; vs2, vacu ole of cell s2. Scale bars, 10 �m .
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146
C h rist en sen , Su b ram an ian , an d D rew s
TABLE 6
Su m m ary of D efect s O bserved in D evelopin g gfa3 Fem ale G am et oph yt es
N o. of abn orm al fem ale gam et oph yt es observed
a
Pist il
st age a
N o. of
pist ils
an alyzed
N o. of
FG s
an alyzed
Fou r n u clei an d
com pressed
N o FG
U n cellu larized
U n fu sed
polar n u clei
T ot al
FG 4/ 5
FG 5
FG 5/ 6
FG 7
4
5
7
18
80
54
78
304
1
5
2
0
0
1
6
18
2
3
3
20
0
1
1
54
3
10
12
92
Assign m en t of a pist il st age is described u n der M at erials an d M et h ods.
fi lled w it h au t ofl u orescen t m at erial. O ccasion ally (6/ 33)
t h e less severe ph en ot ype sh ow n in Fig. 4C w as observed. In
su ch fem ale gam et oph yt es, t h e n u clei w ere presen t in t h e
correct n u m bers an d at t h e appropriat e posit ion s t o be t h e
polar n u clei, egg n u cleu s, an d syn ergid n u clei, bu t a prom in en t cen t ral vacu ole w as absen t (com pare Fig. 4C w it h Fig.
1C ). In som e (3/ 6) of t h ese, t h e “ polar n u clei” w ere adjacen t
t o each ot h er bu t w ere u n fu sed (e.g., t h e fem ale gam et oph yt e sh ow n in Fig. 4C ). G en erally, on e or m ore of t h e
n u clei h ad an irregu lar sh ape an d t h u s w as probably in t h e
process of degen erat ion (dat a n ot sh ow n ). T ak en t oget h er,
t h ese dat a su ggest t h at developm en t of fem 1 fem ale gam et oph yt es begin s t o depart from w ild t ype ju st before
polar n u clei fu sion (lat e st age FG 5) an d t h at an early even t
appears t o be degen erat ion of t h e cen t ral vacu ole.
fem4. At the term inal developm ental stage (stage FG7),
50% of em bryo sacs in fem 4/FEM4 pistils were abnorm al
(Table 5), corresponding to the 100% penetrance of the fem 4
m utation (Table 2). The term inal phenotype of fem 4 fem ale
gam etophytes is shown in Figs. 4D– 4H and depicted in Fig.
2F. Essentially all (85/86) of the fem 4 fem ale gam etophytes we
observed at the term inal developm ental stage had defects in
cellular m orphology in the cells at the em bryo sac’s m icropylar pole (Table 7). In wild type, the egg cell has a distinctive
pear shape and a highly polarized cytoplasm ; its nucleus and
m ost of its cytoplasm are at its chalazal end and a large
vacuole occupies the rem aining three-fourths of the cell (Fig.
1). Likewise, wild-type synergid cells have a distinctive shape
and a highly polarized cytoplasm , which has the opposite
polarity of the egg cell cytoplasm (Fig. 1). As with wild type,
fem 4 fem ale gam etophytes at the term inal developm ental
stage had three cells at the m icropylar pole. However, in
contrast to wild type, these cells had irregular shapes and
polarities. An exam ple of a fem 4 fem ale gam etophyte at the
term inal developm ental stage is shown in Figs. 4D–4F. In this
exam ple, the egg cell is not pear shaped and is tilted relative to
the fem ale gam etophyte’s long axis (Fig. 4E), and the synergid
cells were abnorm ally shaped and their nuclei occupied positions m ore chalazal and abaxial than those of wild type (Figs.
4D and 4F).
In addit ion t o t h e cellu lar m orph ology defect s exh ibit ed
by all fem 4 fem ale gam et oph yt es, som e m at u re fem 4 fe-
TABLE 7
Su m m ary of D efect s O bserved in D evelopin g fem 4 Fem ale G am et oph yt es
N o. of abn orm al fem ale gam et oph yt es observed
Pist il
st age a
N o. of
pist ils
an alyzed
N o. of
FG s
an alyzed
Abn orm al
n u clear
posit ion at
st age FG 4
FG 4
FG 4/ 5
FG 5
FG 5/ 6
FG 6
FG 6/ 7
FG 7
4
4
5
2
2
4
10
55
44
58
32
21
35
172
2
4
3
4
0
0
0
a
b
D evelopm en t al
delay
Abn orm al
cellu lar
m orph ology
at st age FG 5
Abn orm al
cellu lar
m orph ology
at st age FG 6
Abn orm al
cellu lar
m orph ology
at st age FG 7
T ot al
0
0
3b
0
10
0
1
1
2
7
9
0
15
35
1
1
0
0
0
2
23
0
0
0
0
0
0
27
4
7
13
13
10
17
86
Assign m en t of a pist il st age is described u n der M at erials an d M et h ods.
O n e of t h ese fem ale gam et oph yt es w as at st age FG 3, an d t h e ot h ers w ere at st age FG 4.
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147
Fem ale G am et oph y t e M u t an t s
m ale gam et oph yt es also exh ibit ed defect s in polar n u clei
fu sion an d an t ipodal cell degen erat ion , w h ich are developm en t al st eps t h at follow cellu larizat ion (Fig. 2A). O f t h e 86
abn orm al fem ale gam et oph yt es observed at t h e t erm in al
developm en t al st age, 27% h ad in t act an t ipodal cells an d
41% h ad u n fu sed polar n u clei an d in t act an t ipodal cells
(Fig. 4H , T able 7).
T o det erm in e t h e developm en t al st age at w h ich cellu lar
m orph ology becom es abn orm al, w e an alyzed fem ale gam et oph yt e developm en t in fem 4/ FEM 4 pist ils at all developm en t al st ages. T h ese dat a are su m m arized in T able 7. In
w ild t ype, cellu larizat ion begin s im m ediat ely follow in g t h e
t h ird m it osis (early st age FG 5) an d by lat e st age FG 5, t h e egg
an d syn ergid cells h ave dist in ct polarit ies an d m orph ologies
as described above (Fig. 2A; C h rist en sen et al., 1997). In
fem 4 fem ale gam et oph yt es at lat e st age FG 5, t h e egg an d
syn ergid cells already exh ibit ed defect s in cell sh ape, vacu olar sh ape, an d cell posit ion , in dicat in g t h at cellu lar m orph ology w as abn orm al as soon as t h ese cells w ere form ed
(T able 7).
An alysis of fem ale gam et oph yt e developm en t in t h e fem 4
m u t an t also revealed t w o su bt le defect s t h at occu rred
before st age FG 5 an d w ere n ot m an ifest ed at t h e t erm in al
developm en t al st age (T able 7). T h e fi rst w as abn orm al
n u clear posit ion at st age FG 4 (“ Abn orm al n u clear posit ion
at st age FG 4” colu m n in T able 7). In fem 4/ FEM 4 pist ils,
som e fou r-n u cleat e em bryo sacs w ere observed in w h ich
on e of t h e t w o n u clei at each pole w as closer t o t h e cen t er
of t h e em bryo sac t h an in w ild t ype (dat a n ot sh ow n ). T h e
secon d defect w as apparen t developm en t al asyn ch ron y
(“ D evelopm en t al delay” colu m n in T able 7). We previou sly
sh ow ed t h at m egagam et ogen esis in w ild t ype is fairly
syn ch ron ou s an d t h at fem ale gam et oph yt es at st age FG 4 are
n ot presen t in pist ils con t ain in g fem ale gam et oph yt es at
st age FG 6 (C h rist en sen et al., 1997). H ow ever, som e fem 4/
FEM 4 pist ils con t ain ed bot h st age FG 4 an d st age FG 6
fem ale gam et oph yt es (T able 7). N evert h eless, fem 4 fem ale
gam et oph yt es do progress beyon d st age FG 4 becau se by t h e
t erm in al st age n early all (85/ 86) fem 4 fem ale gam et oph yt es
observed h ad u n dergon e t h e t h ird m it osis an d h ad becom e
cellu larized (T able 7).
fem 2, fem 3, gfa4, and gfa5. T h e ph en ot ype of t h e fem 2,
fem 3, gfa4, an d gfa5 m u t an t s is depict ed in Fig. 2C . In t h is
grou p, m egam et ogen esis did n ot progress beyon d st age FG 1
(on e-n u cleat e st age; Fig. 2A). As sh ow n in Fig. 3A, ovu le
m orph ology (in t egu m en t len gt h an d ovu le sh ape) w as sim ilar t o ovu les con t ain in g m at u re, w ild-t ype fem ale gam et oph yt es. H ow ever, fem ale gam et oph yt e m orph ology w as
sim ilar t o fem ale gam et oph yt es at st age FG 1 [t ear drop
sh ape, on e n u cleu s, an d sm all vacu oles (Webb an d G u n n in g, 1990; C h rist en sen et al., 1997)]. We did n ot exam in e
earlier developm en t al st ages in t h is grou p.
Wit h in fem 2/ FEM 2, fem 3/ FEM 3, gfa4/ G FA 4, an d gfa5/
G FA 5 pist ils, t h e frequ en cies of m orph ologically abn orm al fem ale gam et oph yt es w ere 51, 47, 10, an d 18% ,
respect ively. T h e fact t h at t h e t h ese frequ en cies w ere
h igh er t h an expect ed for t h e fem 2 an d fem 3 m u t an t s
(based on t h e gen et ic t ran sm ission dat a in T able 2)
su ggest s t h at t h ese m u t at ion s m ay be lin k ed t o ch rom osom al rearran gem en t s (Ray et al., 1997). T h e fact t h at
t h ese frequ en cies w ere h igh er t h an expect ed for t h e gfa4
an d gfa5 m u t an t s [based on t h e 42% pen et ran ce of t h e
gfa4 m u t at ion an d t h e 79% pen et ran ce of t h e gfa5
m u t at ion report ed by Feldm an n et al. (1997)] m ay su ggest
t h at som e m orph ologically n orm al fem ale gam et oph yt es
w ere n on fu n ct ion al.
Microgam et ogenesis in t he fem and gfa Mut ant s
As sh ow n in T able 1, t h e fem 3, fem 4, gfa2, gfa3, gfa4,
gfa5, an d gfa7 m u t at ion s also affect t h e m ale gam et oph yt e.
T o det erm in e w h et h er t h ey affect m icrogam et ogen esis, w e
an alyzed t h e t erm in al ph en ot ypes of t h ese m u t an t s. T o do
t h is, w e u sed C LSM t o an alyze t h e m ale gam et oph yt es
w it h in an t h ers from lat e st age 12 (ju st prior t o deh iscen ce)
fl ow ers (Sm yt h et al., 1990). T h e resu lt s of t h is an alysis are
sh ow n in Fig. 5. Wit h in fem 3/ FEM 3, gfa3/ G FA 3, gfa4/
G FA 4, an d gfa5/ G FA 5 an t h ers, m orph ologically abn orm al
pollen grain s w ere observed (Figs. 5B–5E). Wit h t h ese m u t an t s, abn orm al pollen grain s w ere collapsed (arrow s) or
h igh ly vacu olat e (arrow h eads). T h ese dat a in dicat e t h at t h e
fem 3, gfa3, gfa4, an d gfa5 m u t at ion s affect m icrogam et ogen esis. By con t rast , on ly m orph ologically n orm al pollen
grain s (over 75 pollen grain s scored) w ere observed in
fem 4/ FEM 4 an d gfa2/ G FA 2 an t h ers (Figs. 5F an d 5G ), su ggest in g t h at t h e fem 4 an d gfa2 m u t at ion s m ay affect som e
aspect of m ale gam et oph yt e fu n ct ion .
D ISCU SSION
Isolat ion of Fem ale G am et op hy t e Mut ant s
M olecu les m ediat in g an d regu lat in g t h e m ajor even t s of
m egagam et ogen esis (m it osis, polarit y est ablish m en t , vacu ole form at ion , n u clear m igrat ion , cellu larizat ion , cell specifi cat ion an d differen t iat ion , n u clear fu sion , an d cell deat h )
an d fem ale gam et oph yt e fu n ct ion (pollen t u be gu idan ce,
fert ilizat ion , in du ct ion of seed developm en t , an d gam et oph yt ic m at ern al con t rol) h ave n ot been iden t ifi ed. T o begin
t o iden t ify t h ese m olecu les, w e h ave in it iat ed a gen et ic
screen for fem ale gam et oph yt e m u t an t s t h at is based on
t w o crit eria, seed set an d segregat ion dist ort ion . Each of
t h ese crit eria alon e is in effect ive in defi n it ively iden t ifyin g
fem ale gam et oph yt e m u t an t s. Redu ced seed set screen s are
lim it ed becau se t h is ph en ot ype also can be cau sed by a
variet y of ot h er fact ors in clu din g adverse en viron m en t al
con dit ion s (e.g., h igh grow t h t em perat u re or w at er st ress),
ch rom osom al rearran gem en t s (e.g., reciprocal t ran slocat ion s an d large in version s), an d sporoph yt ic m u t at ion s (e.g.,
a fem ale-st erile m u t at ion w it h part ial pen et ran ce). Segregat ion dist ort ion screen s are lim it ed becau se t h ey iden t ify
bot h fem ale gam et oph yt e an d m ale gam et oph yt e m u t at ion s. H ow ever, w h en com bin ed, t h ese t w o crit eria are
effect ive in iden t ifyin g fem ale gam et oph yt e m u t an t s. U sin g
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148
C h rist en sen , Su b ram an ian , an d D rew s
FIG. 5. C LSM im ages of m at u re pollen grain s from w ild t ype an d t h e fem 3, fem 4, gfa2, gfa3, gfa4, an d gfa5 m u t an t s. (A) Wild t ype. (B)
fem 3. (C ) gfa3. (D ) gfa4. (E) gfa5. (F) fem 4. (G ) gfa2. Arrow s sh ow collapsed pollen grain s; arrow h ead sh ow s h ypervacu olat e pollen grain . All
im ages are sin gle 1.5-�m opt ical sect ion s. Scale bar, 10 �m .
t h is screen in g procedu re, w e iden t ifi ed fou r lin es (fem 1,
fem 2, fem 3, an d fem 4) con t ain in g fem ale gam et oph yt e
m u t at ion s. Several fem ale gam et oph yt e m u t an t s w ere previou sly iden t ifi ed based on redu ced seed set or segregat ion
dist ort ion in clu din g t h e A rab id opsis ct r1, em b 173, G f,
gfa1 � gfa7, h d d , prl, an d t rp1;t rp4 m u t an t s (Redei, 1965;
C ast le et al., 1993; Kieber et al., 1993; N iyogi et al., 1993;
Sprin ger et al., 1995; Feldm an n et al., 1997; M oore et al.,
1997) an d t h e m aize ig, lo1, lo2, sp1, an d sp2 m u t an t s
(M an glesdorf, 1931; Sin glet on , 1932; Rh oades an d Rh oades,
1939; Sin glet on , 1940; Sin glet on an d M an gelsdorf, 1940;
N elson an d C lary, 1952; Kerm icle, 1971). T h e isolat ion of
t h ese m u t an t s, as w ell as t h e m u t an t s described in t h is
paper, dem on st rat es t h at t h e crit eria of redu ced seed set an d
segregat ion dist ort ion sh ou ld be effect ive in large-scale
screen s for fem ale gam et oph yt e m u t an t s.
Fem ale G am et op hy t e-Sp ecific and G eneral
G am et op hy t ic Mut at ions
T h e m u t an t s discu ssed in t h is paper fall in t o t w o classes.
T h e fem 1 an d fem 2 m u t at ion s affect t h e fem ale gam et o-
ph yt e bu t n ot t h e m ale gam et oph yt e an d, t h u s, fall in t o t h e
fem ale gam et oph yt e-specifi c class. T h e fem 3, fem 4, gfa2,
gfa3, gfa4, gfa5, an d gfa7 m u t at ion s affect bot h t h e fem ale
an d m ale gam et oph yt e an d, t h erefore, are gen eral gam et oph yt ic (Feldm an n et al., 1997). Fem ale gam et oph yt especifi c m u t at ion s are im port an t becau se t h e affect ed gen es
en code fu n ct ion s requ ired by t h e fem ale gam et oph yt e bu t
n ot t h e m ale gam et oph yt e in clu din g t h e est ablish m en t of
fem ale gam et oph yt e polarit y, specifi cat ion an d differen t iat ion of t h e fem ale gam et oph yt e cells, con t rolled cell deat h
of t h e an t ipodal an d syn ergid cells, cen t ral vacu ole form at ion an d m ain t en an ce, pollen t u be gu idan ce, in du ct ion of
seed developm en t , an d gam et oph yt ic m at ern al con t rol
(D rew s et al., 1998). G en eral gam et oph yt ic m u t at ion s are
also an im port an t m u t at ion class becau se m an y im port an t
cellu lar processes are requ ired in bot h gam et oph yt es in clu din g m it osis, vacu ole form at ion , cell expan sion , su bcellu lar m igrat ion , n u clear m igrat ion an d fu sion , an d cellu larizat ion . T h u s, m u t at ion s affect in g som e of t h e m ajor even t s
of m egagam et ogen esis are lik ely t o fall in t o t h e gen eral
gam et oph yt ic class (D rew s et al., 1998). Wit h all of t h e
m u t an t s described in t h is paper, w e do n ot yet k n ow
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149
Fem ale G am et oph y t e M u t an t s
w h et h er t h e correspon din g m u t at ion s also affect t h e sporoph yt e.
T he fem 1, fem 2, fem 3, fem 4, gfa2, gfa3, gfa4, gfa5,
and gfa7 Mut at ions A ffect Megagam et ogenesis
All of t h e fem ale gam et oph yt e m u t at ion s w e an alyzed
affect m egagam et ogen esis. T h ese n in e m u t at ion s affect a
ran ge of st eps. In t h e fem 2, fem 3, gfa4, an d gfa5 m u t an t s,
m egagam et ogen esis does n ot progress beyon d t h e on en u cleat e st age (st age FG 1; Figs. 2C an d 3A). D evelopm en t al
arrest at t h e on e-n u cleat e st age cou ld resu lt from defect s in
a variet y of processes in clu din g m it osis, cell cycle regu lat ion , cellu lar m et abolism , or developm en t al con t rol. From
t h e gen et ic an d m orph ological dat a presen t ed h ere, t h ere is
n o w ay t o det erm in e w h ich of t h ese processes is affect ed by
t h e fem 2, fem 3, gfa4, an d gfa5 m u t at ion s.
T h e gfa2 m u t at ion affect s fu sion of t h e polar n u clei (Figs.
2D an d 3B–3D ). In t h is m u t an t , t h e polar n u clei m igrat e
t oget h er, com e t o lie side-by-side, bu t fail t o fu se. Polar
n u clei fu sion h as been described in plan t s an d h as been
sh ow n t o con sist of t w o st eps. In t h e fi rst st ep, t h e en doplasm ic ret icu lu m (ER) m em bran es t h at are con t in u ou s
w it h t h e ou t er n u clear m em bran es of t h e t w o n u clei fu se,
resu lt in g in a con t in u ou s ou t er m em bran e su rrou n din g
u n fu sed in n er n u clear m em bran es; an d in t h e secon d st ep,
t h e in n er n u clear m em bran es com e in con t act an d m erge t o
com plet e t h e fu sion process (Jen sen , 1964). We do n ot yet
k n ow w h ich of t h ese st eps is affect ed by t h e gfa2 m u t at ion .
N u clear fu sion h as also been st u died in yeast an d h as been
fou n d t o occu r via a process sim ilar t o t h at in plan t s
(Ku rih ara et al., 1994). Yeast n u clear fu sion m u t an t s h ave
been iden t ifi ed an d som e of t h e affect ed gen es en code
com pon en t s of t h e t ran slocon , w h ich is a prot ein com plex
em bedded in t h e ER m em bran e t h at is respon sible for
prot ein t ran slocat ion in t o t h e ER (M arsh an d Rose, 1997).
Polar n u clei fu sion is also affect ed by t h e gfa3 an d gfa7
m u t at ion s (Figs. 2D , 3E, an d 3F). H ow ever, u n lik e t h e gfa2
m u t at ion , t h e gfa3 an d gfa7 m u t at ion s are pleiot ropic an d
also resu lt in u n cellu larized fem ale gam et oph yt es (Figs. 2E,
3G , an d 3I). Pleiot ropy of m u t at ion s affect in g polar n u clei
fu sion is n ot su rprisin g becau se, in yeast , som e of t h e
m olecu les requ ired for n u clear fu sion are also requ ired for
ot h er cellu lar processes an d, as a con sequ en ce, som e of t h e
yeast n u clear fu sion m u t at ion s are pleiot ropic (M arsh an d
Rose, 1997). For exam ple, t h e yeast Sec63 prot ein is requ ired for bot h n u clear fu sion an d prot ein t ran slocat ion
in t o t h e ER lu m en (N g an d Walt er, 1996). T h u s, by an alogy,
t h e pleiot ropy exh ibit ed by t h e gfa3 an d gfa7 m u t at ion s
cou ld refl ect addit ion al fu n ct ion s of t h e G FA 3 an d G FA 7
gen e produ ct s.
fem 1 fem ale gam et oph yt es develop n orm ally u n t il lat e
du rin g st age FG 5 (T able 5), w h ich is ju st before polar n u clei
fu sion bu t aft er t h ree rou n ds of m it osis an d cellu larizat ion .
T h u s, t h e fem 1 m u t at ion does n ot affect m an y fu n dam en t al
cellu lar processes su ch as m it osis, vacu ole form at ion ,
n u clear m igrat ion , an d cellu larizat ion . In som e (6/ 33, T able
5) fem 1 em bryo sacs, m ost aspect s of m orph ology are
n orm al except t h at t h e cen t ral vacu ole is n ot presen t (Fig.
4C ). T h u s, FEM 1 m ay be requ ired for m ain t en an ce of t h e
cen t ral vacu ole an d t h e fem 1 m u t at ion m ay cau se disin t egrat ion of t h is vacu ole. If t h is is t ru e, t h en t h e m ore severe
ph en ot ype observed (Figs. 4A an d 4B) cou ld be cau sed by t h e
release of vacu olar con t en t s. Addit ion al an alysis u sin g
t ran sm ission elect ron m icroscopy is requ ired t o det erm in e
if t h at is t h e case.
fem 4 fem ale gam et oph yt es exh ibit defect s in cellu lar
m orph ology (Figs. 2F an d 4D – 4H ). T h e m ech an ism of
cellu larizat ion du rin g m egagam et ogen esis is n ot w ell u n derst ood (Ru ssell, 1993). O n e m odel is t h at follow in g t h e
t h ird m it osis, m icrot u bu les em an at e radially from in dividu al n u clei, ph ragm oplast s form w h ere t h e m icrot u bu les
m eet , an d cell plat es form at t h e equ at or of t h e ph ragm oplast s (Brow n an d Lem m on , 1991; Ru ssell, 1993). In su pport
of t h is m odel, ph ragm oplast s h ave been observed in A rab id opsis fem ale gam et oph yt es bet w een sist er an d n on sist er
n u clei follow in g t h e t h ird m it osis (Webb an d G u n n in g,
1994). In t h is m odel, t h e locat ion of t h e cell w alls is
u lt im at ely det erm in ed bot h by t h e posit ion of t h e n u clei
an d t h e organ izat ion of t h e cyt osk elet on . If t h is m odel is
correct , t h en t h e fem 4 m u t at ion cou ld affect cellu lar m orph ology by disru pt in g n u clear posit ion or t h e organ izat ion
or fu n ct ion of t h e cyt osk elet on . C yt osk elet al organ izat ion
defect s also cou ld explain t h e aberran t n u clear posit ion an d
apparen t developm en t al delay observed in fem 4 fem ale
gam et oph yt es at st age FG 4 (T able 7). Fu rt h er an alysis of
cellu larizat ion an d cyt osk elet al organ izat ion in fem 4 fem ale gam et oph yt es is requ ired t o address t h ese issu es.
Som e fem 4 em bryo sacs also exh ibit defect s in polar n u clei
fu sion an d an t ipodal cell degen erat ion (T able 7); h ow ever,
becau se t h ese developm en t al st eps occu r aft er cellu larizat ion , t h ese defect s probably occu r as a secon dary con sequ en ce of t h e earlier cellu larizat ion defect .
O f t h e previou sly iden t ifi ed fem ale gam et oph yt e m u t an t s, m orph ological an alysis t h rou gh ou t m egagam et ogen esis h as been carried ou t on ly w it h t h e G f (Redei,
1965), h d d (M oore et al., 1997), ig (Kerm icle, 1971), lo2
(N elson an d C lary, 1952), an d prl (Sprin ger et al., 1995)
m u t at ion s. T h e G f m u t at ion cau ses a ph en ot ype iden t ical
t o t h at of t h e fem 2, fem 3, gfa4, an d gfa5 m u t an t s,
developm en t al arrest at t h e on e n u cleat e st age (st age
FG 1). By con t rast , t h e ph en ot ypes of t h e h d d , ig, lo2, an d
prl m u t an t s do n ot resem ble t h ose of t h e m u t an t s presen t ed h ere. T h e h d d , lo2, an d prl m u t at ion s cau se
developm en t al arrest at t h e on e-, t w o-, fou r-, or eigh t n u cleat e st age (M oore et al., 1997; Sh eridan an d H u an g,
1997), an d t h e ig m u t at ion cau ses ext ra cycles of n u clear
division s t o occu r (Kerm icle, 1971; Lin , 1978; Lin , 1981;
H u an g an d Sh eridan , 1996). In addit ion , t h e h d d an d ig
m u t at ion s cau se asyn ch ron y of n u clear division s at t h e
m icropylar an d ch alazal poles (H u an g an d Sh eridan , 1996;
M oore et al., 1997). T h ese dat a su ggest t h at t h e H D D , IG ,
LO 2, an d PR L gen es m ay play a role in con t rollin g
n u clear division . C on sist en t w it h t h is idea, t h e PR L gen e
C opyrigh t © 1998 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved.
150
C h rist en sen , Su b ram an ian , an d D rew s
is relat ed t o t h e yeast M C M 2-3-5 gen es t h at are requ ired
for in it iat ion of D N A replicat ion (Sprin ger et al., 1995).
T he fem 3, gfa3, gfa4, and gfa5 Mut at ions A ffect
Microgam et ogenesis
As discu ssed above, t h e fem 3, fem 4, gfa2, gfa3, gfa4, gfa5,
an d gfa7 m u t at ion s affect bot h t h e fem ale an d m ale gam et oph yt e. T h u s, in form at ion abou t t h e pollen defect s cau sed by
t h ese m u t at ion s m ay h elp t o u n derst an d t h e fu n ct ion s of
t h e correspon din g gen es du rin g m egagam et ogen esis. As a
fi rst st ep in t h is direct ion , w e an alyzed t h e t erm in al pollen
ph en ot ypes of t h ese m u t an t s. We fou n d t h at fem 3, gfa3 (an d
presu m ably also gfa7), gfa4, an d gfa5 affect m icrogam et ogen esis. Fu rt h er an alysis is requ ired t o det erm in e t h e specifi c st eps of m icrogam et ogen esis affect ed. By con t rast , t h e
gfa2 an d fem 4 m u t at ion s do n ot appear t o affect m icrogam et ogen esis an d m ay in st ead affect som e aspect of pollen
fu n ct ion su ch as pollen germ in at ion , pollen t u be grow t h , or
pollen t u be gu idan ce.
ACKN OWLED GMEN TS
We t h an k Ken Feldm an n for providin g t h e gfa2, gfa3, gfa4, gfa5
an d gfa7 m u t an t s, T om Jack for providin g T -D N A m u t agen ized
lin es, an d t h e A rab id opsis Biological Resou rce C en t er for m ak in g
available t h e Ken Feldm an n collect ion an d m s1-1. We also t h an k
D ian a Lee for h elpfu l com m en t s on t h e m an u script . T h is w ork w as
su pport ed by N at ion al Scien ce Fou n dat ion G ran t IBN -9630371.
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Received for pu blicat ion April 20, 1998
Revised Ju n e 10, 1998
Accept ed Ju n e 10, 1998
C opyrigh t © 1998 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved.