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Protoplasma (1998) 203:1-15 PROTOPL SM9 Sp ringer-Verlag 1998Pr in ted in Aus t r ia

n v i v o s t u d ie s o n t h e n u c l e a r b e h a v i o r o f t h e a r b u s c u l a r m y c o r r h i z a l f u n g u si gaspor a rosea g r o w n u n d e r a x e n i c c o n d i t i o n s

B . B a g o ~ * , W . Z i p f e l 2 , R . M . W i l l i a m s 2 , H . C h a m b e r l a n d 3 , J . G . L a f o n t a i n e 3 , W . W . W e b b 2 , a n d Y . P i c h ~ 11Centre de Reche rche en Biolo gie Foresti6re and 3D@artement de Biolog ie, Universit6 Laval, Ste-Foy , Qudbec and 2Applied and En gineeringPhysics, Cornell University , I thaca, New YorkRec eived January 27, 1998Accepted April 28, 1998

S umma r y The distr ibution and fate of nuclei of the arbuscular-my-corrhizal fungus Gigaspora rosea during late stages of axen ic cultureswere studied in f ixed cultures by tran smitted l ight, convention al andconfo cal laser scanning micro scopy, and in l ive cultures with two-pho ton f luo rescence mic roscopy . Matu re spec imens no t ye t showingapical septation displayed ova l-shaped nuc lei localized in lateralposit ions o f the hypha all a long the germ-tub e length. B eside these,round-shaped nuc le i were found to m igra te a long the cen t ra l ge rm-tube core. So me (rare) germ-tub e areas, delim ited by septa and con-taining irregularly shaped, much brighter f luorescent nuclei werealso found. Specimens that had just init iated the septation processafter germ-tube growth arrest displayed round or oval-shaped nucleiin several portions of the germ tubes. These hy phal areas often alter-nated with other septa-delimited cytoplasmic clusters which con-tained distorted, brightly f luorescen t nuclei . Com pletely septatedspec imens mos t ly lacked nuc le i a long the i r ge rm tubes. How ever ,highly f luorescent chromatin masses appeared within remnants ofcy top lasmic mate r ia l , o f ten compressed be tween c lose sep ta . Ourresults provid e a f irst c lear picture of the in viv o distr ibution ofnuc le i a long a rbuscu la r mycorrh iza l funga l ge rm tubes i s sued f romresting spores, an d suggest that selective areas of their coeno cytichyphae are under specific , s ingle nuclear control. They indicate aswel l tha t random au to ly t ic p rocesses occur a long senesc ing G roseagerm tubes, probably as a consequence of the absence of a host rootsignal for mycorrhizal formation. Finally , the data presented herea l low us to env isage the fa te o f nuc le i re leased by the ge rm ina t ingspore after nonsymbiotic fungal growth arrest .Keyword s : Arbu scular-m ycorrhiza l fungi; Axen ic culture; DA PI;Gigaspora rosea; Mult ipho ton m ic roscopy ; Nuc le i .

*Corres ponden ce and reprints (present address): Plant and Soil Bio-phys ics Labora to ry , Eas te rn Reg iona l Resea rch Cen te r , ARS, U.S .Depar tmen t o f Agr icu l tu re , 600 E . Mermaid Lane , W yndmoor , PA19038, U.S.A.

Abbrev ia t ions: AM fung i a rbuscu la r -mycorrh iza l fung i ; DAP I 4 ' , 6 -d iamid ino-2 -pheny l indo le ; FM f luorescence mic roscopy ; CLSMconfoca l la se r scann ing mic roscopy ; 2PM two-pho ton mic roscopy ;PI p rop id ium iod ide ; PMT pbo tomul t ip l ie r tube .

I n t r o d u c t i o nA r b u s c u l a r my c o r r h i z a l f u n g i A M f u n g i; G lo m a le s ,Z y g o m y c o t a ) c o l o n i z e ro o t s o f m o s t l a n d p la n t s t of o r m a r b u s c u l a r my c o r r h i z a s S mi th an d R e a d 1 9 9 7 ) .T h e e c o lo g i c a l a n d e c o n o mic a l imp o r t a n c e o f t h i ss y m b io s i s h a s l e d t o a n i n c r e a s e d i n t e r e s t i n th e s t u d yo f t h i s b io t r o p h i c a s s o c i a t i o n f r o m w h ic h b o th p a r t -n e r s b e n e f i t A z c 6 n - A g u i l a r an d B a g o 1 9 9 4 , B a r e aa n d J e f f r ie s 1 9 9 5 ) . O n e o f th e ma jo r d i f f i c u l ti e s o fs u c h s t u d i e s i s t h e i n a b i l i ty o f th e A M f u n g i t o f u l f il lt h e i r l if e c y c l e i n t h e a b s e n c e o f a h o s t r o o t , w h ic h h a sl e d t o t h e i r c l a s s i f i c a t i o n a s o b l i g a t e s y mb io n t s

A z c 6 n - A g u i l a r a n d B a r e a 1 9 9 4 ) .W h e n a x e n i c a l l y c u l t u r e d , t h e A M f u n g a l s p o r e g e r -min a t e s a n d i t s t h i c k - w a l l e d g e r m tu b e e x t e n d s i n t h ec u l t u r e me d iu m, o c c a s io n a l l y f o r min g r i g h t - a n g l e db r a n c h e s . U n d e r t h e s e c o n d i t i o n s , d e v e lo p me n t c a nb e m a i n t a i ne d u p t o 2 - 4 w e e k s d e p e n d i n g o n t h e f u n -g a l s p e c i e s , g r o w th me d iu m, a n d s p o r e a g e ) , a n d t h eg e r m tu b e c a n r e a c h s e v e r a l c e n t ime te r s i n l e n g th . I ft h e f u n g u s d o e s n o t f i n d a h o s t r o o t t o c o lo n i z e , a p i c a ls e p t a t i o n a n d c y to p l a s m r e t r a c t i o n h a v e b e e n r e p o r t e dto o c c u r M o s s e 1 9 6 2 , 1 9 8 8 ; H e p p e r 1 9 8 3 ; B o n f a n t ea n d B ia n c io t t o 1 9 9 5 ; B o n f a n t e a n d P e r o t t o 1 9 9 5 ) . A tth e e n d o f th i s p r o c e s s t h e w h o le g e r m tu b e a p p e a r s

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se p t a t e d a n d e mp ty ( H e p p e r 1 9 8 3 , Mo sse 1 9 8 8 ) , a n dth e sp o r e e n te r s a n e w d o r m a n c y s t a te ( G io v a n n e t t i e ta l . 1 9 9 4 , B o n f a n t e a n d B ia n c io t t o 1 9 9 5 ) . T h e c e s sa -t i o n o f g r o w th i s n o t u n d e r s to o d b u t i s p r o b a b ly n o td u e t o t h e e x h a u s t i o n o f a n y e s se n t i a l c o mp o n e n t o fth e p a r e n t sp o r e t h a t i s a b l e t o r e - g e r min a t e s e v e r a lt ime s ( H e p p e r 1 9 8 4 , B a g o 1 9 9 0 ) . S in c e t h e e a r lys tu d i e s b y G e r d e ma n n ( 1 9 5 5 ) a n d Mo sse ( 1 9 5 9 ) , a l la t t e mp t s t o su p p ly t h e f u n g u s a x e n i c a l l y w i th t h e n e c -e s sa r y f a c to r ( s ) t o o v e r c o m e i ts o b l i g a to r y c o n d i -t i o n h a v e f a i l e d , e v e n t h o u g h A M f u n g a l sp o r e s w e r ef o u n d to c o n t a in t h e me ta b o l i c ma c h in e r y n e c e s sa r yto su s t a in v e g e t a t i v e h y p h a l g r o w th ( r e v i e w e d b yH e p p e r 1 9 8 4 , A z c d n - A g u i l a r a n d B a r e a 1 9 9 4 ) . T h er e q u i r e me n t o f a sw i t c h o n s ig n a l b y t h e h o s t p l a n tt o s t a r t t h e f u n g a l me ta b o l i sm a f t e r sy mb io s i s e s t a b -l i sh me n t h a s b e e n p r o p o se d t o e x p l a in t h e f a i l u r e o fA M f u n g i t o g r o w in a f r e e - l i v in g s t a tu s ( B e c a r d a n dPich6 1989, Bag o e t a l. 1996) . The na tu re o f th is

sw i t c h o n s ig n a l st il l r e ma in s u n k n o w n , a s a r e t h ec y to lo g i c a l p r o c e s se s l e a d in g t o t h e c o mp le t e f u n g a lg r o w th a r r e s t w h e n su c h a s i g n a l i s la c k in g .S i m i la r t o o th e r m e m b e r s o f Z y g o m y c o t a , A M f u n g ia r e c o e n o c y t i c a n d mu l t i n u c l e a t e d ( B ia n c io t t o a n dB o n f a n t e 1 9 9 2 a n d r e f e r e n c e s t h e r e in ) . D i f f e r e n tc y to lo g i c a l s t u d i e s h a v e b e e n c a r r i e d o u t o n t h en u c l e a r s t a tu s o f A M f u n g a l sp o r e s ( S w a r d 1 9 8 1 a , b ;C o o k e e t a l. 1 9 8 7 ; B u r g g r a a f a n d B e r in g e r 1 9 8 9 ;B ia n c io t t o a n d B o n f a n t e 1 9 9 2 ; B 6 c a r d a n d P f e f f e r1 9 9 3 ) a n d h y p h a e , b o th b e f o r e ( S w a r d 1 9 8 1 c , C o o k ee t a l. 1987, Meie r and Charva t 1992, B ianc io t to an dB o n f a n t e 1 9 9 3 ) a n d a f t e r ( B o n f a n t e - F a so lo e t a l.1987, B ianc io t to and Bonfante 1992, Ba les t r in i e t a l .1 9 9 2 ) s y m b i o s i s e st a b li s h m e n t . D o r m a n t A M f u n g a lsp o r e s h a v e b e e n e s t ima te d t o c o n t a in b e tw e e n 1 , 7 0 0to 2 0 , 0 0 0 n u c l e i , d e p e n d in g o n t h e fu n g a l sp e c i e s a n dth e q u a n t i f i c a t io n me th o d u se d ( C o o k e e t a l. 1 9 8 7 ,B u r g g r a a f a n d B e r in g e r 1 9 8 9 , B 6 c a r d a n d P f e f f e r1 9 9 3 ) . W h e n th e sp o r e g e r min a t e s , n u c l e i mig r a t ein to t h e e me r g in g g e r m tu b e ( S w a r d 1 9 8 1 b ) . A c t iv e -ly - g r o w in g g e r m tu b e s t y p i c a l l y d o n o t c o n t a in n u c l e i5 - 2 0 g m f r o m th e a p e x ( S w a r d 1 9 8 1 c , C o o k e e t a l.1 9 8 7 ) . A lo n g th e r e ma in in g h y p h a t h e n u c l e i d i s -p l a y r o u n d o r o v a l sh a p e s , d i sp e r se d c h r o ma t in a n d ac o n sp i c u o u s n u c l e o lu s ( B ia n c io t t o a n d B o n f a n t e1 9 9 2 ) . O b se r v a t i o n s o n f i x e d sp e c ime n s i n d i c a t e dth a t n u c l e i w e r e l i n e a r ly d i s t r i b u t e d a t a c o n s t a n t d e n -s i t y o f a p p r o x . 4 6 0 / c m ( B 6 c a r d a n d P f e f f e r 1 9 9 3 ) ,a p p e a r in g f r e q u e n t ly a s so c i a t e d i n g r o u p s o f 2 o r 3 - 4( C o o k e e t a l. 1 9 8 7 , B ia n c io t t o a n d B o n f a n t e 1 9 9 3 ).P r e v i o u s l y i t h a s b e e n r e p o r t e d t h at A M f u n g i d o n o t

B B a g o e t a l : I n v i v o s t u d ie s o n n u c l e i o f a x e n i c igaspora roseas y n t h e s i z e D N A i n t h e a b s e n c e o f A M f o r m a t i o n( B u r g g r a a f a n d B e r in g e r 1 9 8 9 , V ie r a a n d G le n n1 9 9 0 ) . Mo r e r e c e n t ly , h o w e v e r , D N A sy n th e s i s h a sb e e n c o n f i r m e d b y b r o m o d e o x y u r i d i n e i n c o r p o r a ti o nt e s t s ( B ia n c io t t o a n d B o n f a n t e 1 9 9 3 ) . R e su l t so b t a i n e d w i t h f l u o r e s c e n c e m i c r o s c o p y a n d i m a g ea n a ly se s a l so l e d B 6 c a r d a n d P f e f f e r ( 1 9 9 3 ) t o c o n -c lu d e t h a t n u c l e a r d iv i s io n a n d D N A r e p l i c a t i o n d oo c c u r n o n s y m b i o t i c a ll y i n A M f u n gi .A p a r t f r o m th e a b o v e o b se r v a t i o n s , l i t t l e i s k n o w na b o u t t h e a c tu a l r o l e a n d b e h a v io r o f t h e so me 8 0 0n u c l e i r e l e a se d f r o m th e A M f u n g a l sp o r e ( B 6 c a r da n d P f e f f e r 1 9 9 3 ) a f t e r g e rmin a t i o n . T h i s i s sp e c i a l l yt r u e w h e n c o n s id e r in g t h e f i n a l s t a g e s o f a x e n i cd e v e l o p m e n t , w h e n t h e f u n g u s h a s p r o v e d u n a b l e t of in d a h o s t r o o t to e s t a b l i sh sy m b io s i s w i th a n d s to p sg r o w in g . T h e c u r r e n t k n o w le d g e o n t h i s p a r t i c u l a ri s s u e c o u l d b e s u m m a r i z e d b y t h e s e t w o o b s e r v at i o n s:( i ) Mo sse ( 1 9 8 8 ) , i n a r e v i e w o n d i f f e r e n t a sp e c t s o ft h e i n d e p e n d e n t g r o w t h o f A M f u n g i, p r e s e n t e d t w op h o to g r a p h s o f ea r l ie r u n p u b l i sh e d r e su l t s in w h ic ha c e to - c a r min e s t a in e d n u c l e i o f o ld lomus mosseaea x e n i c h y p h a e d i sp l a y e d d i s to r t e d sh a p e s c o mp a r e dto n u c l e i i n y o u n g , s imi l a r l y s t a in e d g e r m tu b e s ; ( i i )C o o k e e t a l. ( 1 9 8 7 ) r e p o r t e d t h a t o ld , s e p t a t e d h y p h a esh o w e d l i t tl e o r n o c e l l u l a r c o n t e n t a n d l a c k e d n u c l e i .H o w e v e r , s e v e r a l f u n d a me n ta l q u e s t i o n s s t i l l r e ma inu n a n sw e r e d : H o w d o n u c l e i d i s t ri b u t e a lo n g , an d c o n -t r o l t h e l o n g , c o e n o c y t i c f u n g a l g e r m tu b e ? W h a t i st h e i r b e h a v io r a f t e r a s se s sme n t o f sy mb io s i s f a i l u r e ?W h a t is t h e i r f i n a l f a te : a r e t h e y r e a b so r b e d t h r o u g hc y to p l a smic r e t r a c t i o n b y t h e sp o r e w h ic h r e l e a se dth e m, o r , o n t h e c o n t r a r y , d i s c a r d e d a n d d e s t r o y e d ?T h e a n sw e r t o t h e se q u e s t i o n s c o u ld f u r th e r c l a r i f yth e me c h a n i sm s l e a d in g t o f u n g a l g r o w th a r r e s t u n d e ra x e n i c c o n d i t i o n s .T h e r e c e n t d e v e l o p m e n t o f m u l t i - p h o t o n m i c r o s c o p y(Den k e t al . 1990, 1995; Wil l iam s e t a l. 199 4; Xue t a l. 1 9 9 6 ; X u a n d W e b b 1 9 9 6 ) h a s o p e n e d n e wp r o sp e c t s i n c e l l - c y c l e s t u d i e s o n l i v in g sp e c ime n s .D u e t o t h e r e la t i v el y l o w e r l e v e l o f d a m a g e i n d u c e db y th i s t e c h n iq u e o n l i v in g t i s su e s , e sp e c i a l l y w h e nu s in g U V - a b so r b in g f l u o r o p h o r e s , e x p e r ime n t s c a nn o w b e c o n d u c t e d o v e r l o n g e r p e r i o d s o f t i m e(Wil l iam s e t a l. 199 4, Den k e t a l. 1995) . The a im o fth e p r e se n t w o r k w a s t o s t u d y t h e b e h a v io r o f n u c l e io f th e A M f u n g u s igaspora rosea i n v iv o d u r in g i t sn o n s y m b i o t i c p h a s e b y c o m b i n i n g t h e u s e o f th e U V -a b s o r b i n g f l u o r o c h r o m e 4 ' , 6 - d i a m i d i n o - 2 - p h e n y l i n -d o l e ( D A P I ) w i t h 2 - p h o t o n m i c r o s c o p y ( 2 P M ) . T h r e epa r t icu la r po in ts wi l l be addressed: ( i ) nuc lea r d is t r ib -

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B. Bago et al . : In vivo studies on nuclei of axenic Gigaspora roseau t i o n a l o n g t h e g r o w i n g g e r m t u b e s ; ( i i) b e h a v i o r o ff u n g a l n u c l e i d u r in g a x e n i c g r o w t h c e s s a t io n p r o c e s s -e s ; ( i i) f at e o f r e l e a s e d n u c l e i a f t e r f a i l u r e o f A M s y m -b i o s i s e s t a b l i s h m e n t .

a t e r i al a n d m e t h o d sBiological materialSpores o f Gigaspora rosea Nico lson & Schenk ( fo rmer ly cons ide redG. margarita Bago e t a l . 199 8) (DA OM 194757 , B iosys temat icResearch Centre, Ottawa, Canada) were collected from pot culturesby the we t s iev ing and decan ta tion techn ique (Gerdemann and Nico l -son 1963) and surface-steril ized (Mosse 1962).Observations on fi xed specimensSpores were cultured (25 ~ in the dark) in petri dishes (9 cm diam -e~ter) containing 20 ml of an autoclaved (121 ~ 20 rain) water-a garmed ium (0 .8% Bac to -Difco agar in d is ti l led wa ter ) . A m in imum of 5days a f te r ge rmina t ion , ten spec imens were f ixed (10% fo rmal in indisti l led water, 1 h) and care fully transferred with a Pasteur pipette tomicroscope slides. Specimens were then stained with propidiumiodide (PI; Sigma, St. Louis, Mo. , U.S.A.; 5 ~tg/ml), or DAPI (Sig-ma; 25 ~tg/ml) (Heath, 1987). Observ ations by conve ntional f luores-cence mic roscopy (FM) were ca r r ied ou t wi th a Re icher t Po lyvarinstrument, using either blue (PI) or UV (DA PI) i l lumination. Obser-va t ions by la se r confoca l scann ing mic roscopy (CLSM) were pe r -fo rmed e i the r wi th a Ze iss LSM -310 ins t rument (X 100 o i l immer-sion objective; z-axis step, 0 .55 ~m), using an argon laser (514 nm)on spec imens s ta ined wi th P I ; o r wi th an Olym pus IMT -2 ins t rument(DA po 100UV/340, 1 .3 N.A . , 100 oil imm ersion objective; z-axisstep, 0 .3 ~tm) on specim ens stained with DA PI.Observations on in vivo specimensEffec t o f DAPI on spore ge rmina t ion and hypha l deve lopmentTo f ind the mos t innocuous D API concen t ra t ion fo r in v ivo mic ro -scopic studies on G. rosea preliminary viabili ty tests were carriedou t. W ate r -agar med ia con ta in ing d i f fe ren t concen t rat ions o f DA PIwere prepared. The fluor ochrom e was first prepared as a stock solu-tion (1 rag/m1 in disti l led water) , then steril ized by fi l tration (Uniflo13 ram, 0.2 gm pore; Schleicher and Schuell , Keene, N.H. , U.S.A.) .Dif fe ren t vo lumes o f the s te r i le s tock so lu t ion were added to theautoclave d (121 ~ 20 rain) water-aga r med ium to obtain f inal f luo-rochrom e concentrations of 0 , 5 , 12.5, and 25 ~tg/ml. Eight surface-steril ized spores per treatm ent were transferred to petri dishes (9 cmdiamete r ) con ta in ing 20 ml o f the d i f fe ren t med ia. Spores weremaintaine d in culture for 21 days at 25 ~ in the dark, and in a 2%CO2 atmosphere to enhance hyp hal growth (B6card and Pich6 1989).Hypha l deve lopment was measured a t the end o f the assay asdescribed by Marsh (1971) with a 2 mm side grid.T w o - p h o t o n m i c r o sc o p ySpores of G. rosea were pre-germ inated (25 ~ darkness) in water-agar mediu m. Germ inated spores were transferred to special micr o-scope obse rva t ion chambers (chambered coverg lass ; NUNCInterMed, Nap erville , I l l . , U.S.A .) consisting of 75 25 mm N o. 1borosil icate coverglasses bonded to cham bered upper structures withp las t ic removab le l id s . The cham bers w ere f i l led wi th 5 ml o f au to -c laved wa te r -agar med ium am mended wi th 5 ~ tg o f DAP I pe r ml ( f i l -tration-sterilized). This concentration was chose n follow ing the pre-

limina ry viabili ty tests (see Results) . Three pre-ge rminate d sporeswere transferred to each cham bered co verglass, a total of f ive repli-cates were prepared. Spores were cultured at 25 ~ in the dark for 10days, in a sl ight slope posit ion to allow G. rosea germ tubes, whichpresent a negativ e geotropism (Watrud et al . 1978), to grow as closeas poss ib le to the coverg lass . The chambered coverg lass a l lowed thed i rec t, in v ivo obse rva t ion o f G. rosea spec imens a t h igh magn i f ica -tion.Images o f DAPI-s ta ined G rosea were acqu i red wi th a two-pho tonlase r scann ing m ic roscope . 740 nm m ode- locked exc i ta t ion (80 fspu lses , 80 MH z repe t i t ion ra te ) w as ob ta ined f rom an a rgon-pumpedTi:Sapp hire laser (Tsunami, Spectra Physics, Mountain V iew, Calif . ,U.S.A .) . The beam was attenuated and directed into a retrofit ted Bio-Rad MR C-600 confoca l scann ing box a l igned to an inverted mic ro -scope (Zeiss Axiover t -35) wi th a 1 .2 N.A. wa te r immers ionob jec t ive (Zeiss ) . T yp ica l i l lumina t ion powers a t the spec im en were5-10 roW.Because m ul t ipho ton f luo rescence exc i ta t ion i s in t r ins ica l ly loca l -ized (D enk et al. 1990), con focal detection op tics are unnecessary.To avo id s igna l degrada t ion by ex t raneous op t ica l e lemen ts , f luo res -cence de tec t ion was accom pl ished ex te rna l to the confoca l box . T hefluoresc ence was separated from the excita tion l ight by a 680 rim,long-pass dicbroic (Chroma Technology Corp. , Bratt leboro, Vt. ,U.S .A. ) , spec t ral ly f i l te red wi th a 2 mm th ick BGG 22 op t ica l f i lte r(380-530nm) and moni to red by a pho tomul t ip l ie r tube (PMT;Hamamatsu, Bridgew ater, N.J., U.S.A.; HC 125-02) placed conju-gate to the back aperture o f the objective. The signal from the exter-nal PM T is sent to the external input of the MR C-600. The transmit-ted l igh t images-were acqu i red wi th a l igh t p ipe tha t de l ive red l igh tthrough the spe cimen to the standard PM T's in the Bio-Rad confoc albox . Each t ime- lapse se r ies o f images cons is ted o f mul t ip le (1 s )f rames acqu i red a t 30 s in te rvals us ing the m acro p rogramm inglanguage available with the Bio-Rad confocal software. Nuclearm o v e m e n t m o v i e s ho w n a t th e I nt e r n e t' s A M F w e b p ag e(h t tp :/ /www.rsvs .u lava l. ca / -c rb f /amf .h tml) were p repared us ing theAdobe Premiere software.R e s u l t sOb s e r v a t i o n s o n f i x e d s p e c i me n sB o t h P I a n d D A P I s t a in i n g o n f ix e d s p e c i m e n sr e v e a l e d s i m i l a r f e a t u r e s o f s h a p e a n d d i s t r i b u t i o n o fn u c l e i a l o n g f u n g a l h y p h a e , s o t h a t t h e s e r e s u l ts a r ep r e s e n t e d i n d e p e n d e n t l y o f t h e f l u o r o c h r o m e u s e d .C o n v e n t i o n a l F M a n d C L S M o n G. rosea g e r m t u b e sc o n f i r m e d e a r l i e r o b s e r v a t i o n s r e p o r t i n g t h e c o e n o -c y t i c an d m u l t i n u c l e a t e n a t u r e o f t h is A M f u n g u s( F i g . 1 ). N u c l e i d i s t r i b u t e d l i n e a r l y a l o n g h y p h a e , b u tw e r e a b s e n t f r o m t h e a p e x ( F i g. 1 a ) . T h e l a t t e r p o r -t i o n d e v o i d o f n u c l e i w a s g e n e r a l l y l e s s th a n 1 0 ~ tm i nl e n g t h , s o m e t i m e s e x t e n d i n g t o as m u c h a s 5 0 ~ m .N u c l e i i n t h e im m e d i a t e v i c i n i ty o f n o n s e p t a t e da p i c e s w e r e u s u a l l y r o u n d i s h t o o v a l i n s h a p e , a n df o r m e d g r o u p s o f t w o , t h re e , o r f o u r. C L S M r e v e a l e dn u c l e i a s q u i t e h e t e r o g e n o u s l y s t a i n e d , o f te n d i s p l a y -i n g a d a r k e r, c e n t r a l z o n e c o r r e s p o n d i n g t o t h e n u c l e -o l u s ( F i g . 1 a ) .

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Fig. 1 a -d. Conventional fluorescence microscopy on fixed Gigaspora rosea specimens, a Apical zone of a 5-day-old germ tube stained withPI. N ote the hetereogenously stained chromatin and the apparent nucleoli (arrowheads). b and e Nuclear features displayed by germ tubes cen-tral and distal portions (DA PI staining). Striking elongated and irregularly shaped nuclei (double arrowheads) coexist with more regular, roundto oval nuclei (solid arrowheads). Both nuclear types appear in well-delimited hyphal portions, d Higher magnificationof the hyphal branchingportion shown in e. C hromatin strands (double arrowheads) extend severa l microm eters within the hypha . Bars: a, b, and d, 10 hum;c, 20 um

C e n t r a l a n d d i s t a l p o r t io n s o f h y p h a e we re a l s o m u l t i -nuc le a ted (F ig . 1 b , c ) . Bes id es the round is h o r ova ls h a p e s h o we d b y n u c l e i i n s o m e h y p h a l p o r t i o n s , o t h -e r h y p h a l z o n e s d i s p l a y e d n u c l e i w i t h i r r e g u l a r o u t -l ines (F ig . 1 b ) , as wel l as s t rands o f f luo r esce n t ch ro -m a t i n (F i g . 1 c ) . Op t i c a l s e c t i o n s (C L S M , z - s e r i e s ) o fs u c h c h ro m a t i n s t r a n d s c o n f i rm e d t h a t t h e y e x t e n d e ds e v e ra l m i c ro m e t e r s w i t h i n t h e g e rm t u b e (F i g . 1 d) .Observations on in vivo specimensE f f e c t o f D A P I o n s p o r e g e r m i n a t i o n a n dh y p h a l d e v e l o p m e n tR e s u l t s o n t h e e f f e c t o f DA P I o n s p o re g e rm i n a t i o na n d g e rm - t u b e d e v e l o p m e n t a r e s h o wn i n T a b l e 1 .DAP I d i d n o t i n f l u e n c e s p o re g e rm i n a t i o n a t a n y o f

Table 1. Effect of D API on spore germination and hyphaI develop-ment of 21-day-old axenically cultured Gigaspora rosea sporesDAPI 0xg/ml)0.0 5.0 12.5 25.0

Spore germination ( ) 62.5 a 50.0a 75.0a 62.5aHyph al developm ent 753a 485ab 466b 313b(no. of intersections)1Different letters expre ss significant differe nces for P < 0.05 (Z sta-tistic)

t h e c o n c e n t r a t i o n s t e s t e d . Ho we v e r , a s i g n i f i c a n t n e g -a t i v e e f f e c t o n g e r m - t u b e d e v e l o p m e n t w a s f o u n d a tconcen t ra t ions o f 12 .5 and 25 ~ tg /ml . At lower con-c e n t r a t i o n (5 g g / m l ) , s p e c i m e n s d i d n o t s h o w s i g n i f i -c a n t d i ff e r e n c es i n g e r m - t u b e d e v e l o p m e n t c o m p a r e dt o t h e n o n t r e a t e d s p e c i m e n s . T h i s f l u o r0 c h ro m e c o n -c e n t r a t i o n i s w i t h i n t h e r e c o m m e n d e d r a n g e fo r v i t a ls t a i n i n g w i t h DAP I i n c u l t u r e m e d i u m (He a t h 1 9 8 7 ) ,a n d wa s t h e re fo re s e l e c t e d t o c a r ry o u t t h e i n v i v oe x p e r i m e n t s .T w o - p h o t on m i c r o s c o p yT h r e e G rosea s p e c i m e n s (A , B , a n d C ) , r e p re s e n t a -t i v e o f t h e d i f f e r e n t d e v e l o p m e n t a l s t a g e s o f a x e n i c a l -l y c u l t u r e d g e rm t u b e s a r e p r e s e n t e d . T h e r e s t ( 1 2 ) o ft h e s p e c i m e n s s t u d i e d s h o we d f e a t u re s s i m i l a r t o o n eo f t h e s e t h r e e .S p e c i m e n A (F i g s. 2 a n d 3 ) c o n s i st e d o f a n a x e n i c a l -l y c u l t u r e d G rosea s p o r e w h o s e g e r m - t u b e d i d n o ty e t d i s p l a y a p ic a l s e p t a ti o n . A s c h e m a t i c r e p re s e n t a -t i o n o f t h e n u c l e a r d i s t r i b u t i o n a n d f e a t u re s o f t h i ss p e c i m e n i s s h o wn i n F ig . 2 w i t h t h e d i f f e r e n t s t u d i e dz o n e s f r a m e d a n d l a b e l e d . T ra n s m i t t e d - l i g h t ( l e f t) a n d2 P M ( r ig h t ) p i c t u r e s o f t h e c o r r e s p o n d i n g r e g i o n s a r ei l lu s t ra ted in F ig . 3 a - f .S p e c i m e n A d i d n o t s h o w s e p t a t i o n o r c y t o p l a s m i cre t rac t ion in i t s apex (F igs . 2 a and 3 a ) , where the

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a m f . h t m l d i s t al z o n e QFungal - gsporel " " " ' m n f . h l m l " c l o s e t o f - 9 " " " . "" , t h ' e s p o r e " z o n e , ~, ' . - ' ' ' b

' . " , l ) i f/ i ~ r e n t c y t o p l a s q a i c " '" .. ' . . a p p e a r e n c e z o n 6 . . - "f l

A ~ a m f . h lm l a p i ca l z o n e

Fig. 2. Schematic representation of nuclear distribution and features along specimen A germ tubes, rec onstructed from data obtained by 2PM.Black nuclei represent dim, round to ova l-shaped nuclei. Grey nuclei re present irregularly shaped nuclei displaying a brighter fluorescence, a fDetails correspond ing to Fig. 3 a-f . amf.html Time series presented at the AMF web page showi ng nuclear movement along germ tubes. Theschema is not to scale

fungal cytoplasm appeared quite vacuolated (Fig. 3 a,left). 2PM revealed (Fig. 3 a, right) groups of 2-4roundish-oval nuclei at the subapical zone (35 ~tmback the apex), occupying either a central or a lateralposition within the hypha. Time lapse series of thisapical zone (AMF web page) revealed an extremelyslow, if any, cytoplasmic streaming, in which somenuclei seemed to move back from the apex towardsthe spore. These features were maintained along thegerm tube back to the spore to the point where thefirst ramification occurred (Fig. 2); from this pointback to the spore, a more intense cytoplasmic stream-ing was noted.Several millimeters back from the first ramificationand towards the spore, the principal germ tube rantogether with another hypha originating from thesame spore (Figs. 2 b and 3 b). It was not possible toassess if the second hypha was a ramification of theprincipal germ tube or another germ tube issued froma spore regermination. Both hyphae ran very closetogether for several millimeters and at some pointstheir cell walls seemed to merge (Fig. 3 b, left). How-ever, 2PM revealed that, as far as nuclei were con-cerned, each hypha maintained its integrity (Fig. 3 b,right)9 Nuclei appeared round or oval in shape, the lat-ter usually being close to the hyphal wall, whereas the

round nuclei appeared at more central locations. Timelapse series of this and other central zones (AMF webpage) revealed that oval, laterally located nuclei didnot usually move, whereas the more roundish onesmoved along the central part of the germ tube.Nuclear movement seemed to involve cytoplasmicstreaming, and depending on the germ-tube zoneobserved they progressed either towards the apex ortowards the spore.Closer to the spore a ramification containing a groupof auxiliary cells was found (Figs. 2 and 3 c-f).Nuclei within the ramification (the auxiliary cells'subtending hypha), within the auxiliary cells and alsowithin a restricted portion of the principal germ tube(in the proximity of the auxiliary cells' subtendinghypha) were markedly brighter than the nucleidescribed above (Fig. 3 c). While most nuclei wereroundish, a few others appeared irregularly shaped.Nuclei of auxiliary cells were round and bright (Fig. 3d), some irregular, brightly fluorescent strands ofchromatin appeared in auxiliary cells' stems (Fig.3 d). Such chromatin strands were also present at thelower (Fig. 3 c) and upper (Fig. 3 e) parts of the aux-iliary cells' subtending hypha. The apex of this sub-tending hypha was the only one in the whole speci-men A which was septated (Fig. 3e, left). Adjacent to

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~ Fungal ~ t~sp ore ~ / /

Fig. 4. Schem atic representation of specimen B. B lack and gre y nuclei represent nuclei with sim ilar features as in Fig. 2. a j Details corre-sponding to Fig. 5 a -j. T he schem a is not to scale

t h e s e p t a t e d z o n e , t h r e e r o u n d i s h , d i m n u c l e i w e r eo b s e r v e d ( F i g . 3 e , r ig h t ) , f o l l o w e d b y r o u n d b r i g h tn u c l e i , a s w e l l a s o f c h r o m a t i n s t ra n d s ,I n i t i a l l y n o s e p t a w e r e s e e n i n s p e c i m e n A a l o n g t h ep r i n c ip a l g e r m t ub e . H o w e v e r i n t he r e g i o n w h e r e t h ea u x i l i a r y c e l ls ' s u b t e n d i n g h y p h a a r o s e a s i g n i f i c a n td i f fe r e n c e in c y t o p l a s m a p p e a r e n c e w a s n o t e d ( d e li m -i t e d i n F i g s . 2 a n d 3 c , f , le f t ) . W i t h i n t h i s r e g i o n , n e i -t h e r c y t o p l a s m i c s t r e a m i n g n o r n u c l e a r m o v e m e n tw a s f o u n d , b u t t h e n u c l e i e x h i b i t e d b r i g h t e r D A P If l u o r e s c e n c e a n d s o m e o f t h e m w e r e i r r e g u l a r an de v e n s t r a n d - s h a p e d ( F ig . 3 f , r i gh t ) . A p p r o x i m a t e l y3 h a f t e r t h is i n it i a l o b s e r v a t i o n t w o s e p t a f o r m e d ,c l u s t e r i n g th i s d i s t in c t g e r m - t u b e r e g i o n ( n o t s h o w n ) .I m m e d i a t e l y b e h i n d t h i s r e g i o n t h e c y t o p l a s m d i s -p l a y e d a g a i n i t s n o r m a l a p p e a r a n c e ( F ig . 3 f , le f t ) a n dd i m m e r , r o u n d i s h n u c l e i w e r e f o u n d ( F i g. 3 f , r i g h t) .T h e s e f e a t u r e s w e r e m a i n t a i n e d f r o m t h i s r e g i o n t ot h e s p o r e ' s c l o s e s t g e r m - t u b e z o n e s , w h e r e c y t o p l a s -m i c a n d n u c l e a r m o v e m e n t w e r e f a s t e r th a n p r e v i o u s -l y o b s e r v e d ( A M F w e b p a g e ) .S p e c i m e n B ( F i g s . 4 a n d 5 ) c o n s i s t e d o f a G. roseag e r m t u b e w h o s e a p e x h a d i n i t i a t e d s e p t a t i o n . I n t h es c h e m a t i c r e p r e s e n t a t i o n o f th e s p e c i m e n ( F ig . 4 ) th e

d i f f e r e n t z o n e s s t u d i e d a r e f r a m e d a n d l a b e l e d . T h ec o r r e s p o n d i n g t r a n s m i t t e d - l i g h t a n d 2 P M p i c t u r e s a r es h o w n i n F i g . 5 .O b s e r v a t i o n s o n t h i s s p e c i m e n r e v e a l e d s e p t a t e d a p i -c a l z o n e s ( F i g s . 4 a n d 5 a ) s h o w i n g s i m i l a r f e a t u r e s t oh y p h a l z o n e s d e s c r i b e d b y d i f f e r e n t a u t h o r s ( s e eI n t ro d u c t i o n ) a s d e v o i d o f c y t o p l a s m ( f r o m n o w o n ,

c l e a n s e p t a z o n e s ) . N u c l e i w e r e s e e n a s s o o n a sc y t o p l a s m w a s p r e s e n t i n t h e g e r m t u b e ( F i g . 5 a ,r i gh t ) . A l t h o u g h s o m e o f t h e s e n u c l e i w e r e r o u n d i s h ,m o s t o f t h e m a p p e a r e d i rr e gu l ar . I n d e p e n d e n t l y o ft h e i r s h a p e , a l l o f t h e m d i s p l a y e d b r i g h t f l u o r e s c e n c e .T h e s e n u c l e a r f e a t u r e s w e r e m a i n t a in e d t h r o u g h o u t ag e r m - t u b e r e g i o n ( F i g s . 4 a n d 5 a - d ) a l o n g w h i c hc y t o p l a s m a p p e a r e d v a c u o l a t e d a n d / o r g r a nu l ar . N e i -t h e r c y t o p l a s m i c s t r e a m i n g n o r n u c l e a r m o v e m e n tw a s n o t e d . B r i g h t l y f l u o r e s c e n t c h r o m a t i n s t r a n d sw e r e o f t e n o b s e r v e d .A z o n e d i s p l a y i n g c l e a n s e p t a ( F i gs . 4 a n d 5 d ,u p p e r ) m a r k e d t h e e n d o f t h e c y t o p l a s m i c a r e ad e s c r i b e d a b o v e . J u s t b e f o r e t h e f i r s t s e p t u m o f th i sz o n e , a h ig h l y f l u o r e s c e n t m a s s w a s o b s e r v e d ( F i g.5 d , l o w e r ) . Z - s e r i e s o f t h e r e g i o n r e v e a l e d t h a t t hi sm a s s w a s c o n s t i t u te d m a i n l y o f c h r o m a t i n s t r an d s

F ig 3 a-f. In viv o, 2PM of a nonseptated, axenicallyculturedG. rosea specimen (specimenA). Transm itted-light left) and 2PM (right) micro-graphs display cytoplasmicand nu clear features of differentgerm-tubezones, a Apical zone. b Central portion of two close-runninggerm tubes.In ce rtain points cell wails seemed o m erge (solid arrowheads), howev er,nuclei of both hyphae remained ndependent, e Germ-tubezone fromwhich aux iliarycells originate. Both g erm-tube and auxiliarycells' subtending hyphae display bright fluorescentnuclei, usuallyround in shape(solid arrowhead). Some bright chrom atin strands are also noted (double arrowheads), d Higher magnification of the a uxiliary cells showingtheir round, bright nuclei. Some chromatin strands are also e vident n the auxiliarycells' stem (double arrowheads), e Apical zone of the auxil-iary cells' subtendinghyphae. Three round, dim nuclei (solid arrowhead) are followedby brighter rregularnuclei and chromatin strands (dou-ble arrowheads), f Along seve ral microme tersof the germ-tube zone from which auxil iarycells originated a change in cytoplasm appearancewas noted (to the black arrow head's fight) corresponding o brightly fluorescent, rregularlyshaped nuclei n the fluorescent m age (to the whitearrowhead's right). Out of this zone nuc lei remained dimm er and rounder (to the white arrowhead's left). Bars: 20 ~tm

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B. Bago et al.: In vivo studies on nuclei of axenic Gigaspora rosea 9

Fig. 5 a-j. In vivo 2PM of a partially septated, axenically cultured G rosea specimen (specimen B). Transmitted-light (left) and 2PM (right)micrographs display cytoplasmic and nuclear features of different germ-tube zones, a Germ-tube apical zone displaying clean septa and,backwards, granularly vacuolated cytoplasm containing irregularly shaped, brightly f luorescent nuclei, b -d These cytological features are m ain-tained some hundreds of micrometers along the hypha ( towards the spore) (b and e) up to another clean-septa zone (d) . e Higher magn ificat ionof the chromatin mass shown in d (double arrowheads) reveals i ts s trand-like, part iculate appearance, f After the clean-septa zone show n in d,a new cytoplasmic zone containing round, dim nuclei was found, g These features were maintained several hundreds of micrometers backtowards the spore, up to a short clean-septa zone, af ter which a new cytoplasmic zone containing bright , i rregularly shaped nuclei reappeared(double arrowheads) , h -j Zones con taining round, dim nuclei (solid arrowheads) al ternate with others containing irregular , brightly f luorescentnuclei (double arrowheads) along the rest of the germ tube up to the spore. Both cytoplasmic zones are separated from each other by septa. Bars:a-d and f-j , 10 btm; e, 5 ~*m

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10 B. Bago et al.: In vivo studies on nuclei of axenic Gigaspora rosea

Fig. 6 a- d. In vivo microscopy of a fully septated, axenically cultured G rosea specim en (specimen C). Transmitted-light (left) and 2PM (right)micrographs display cytoplasmic and nuclear features of different germ-tube zones, a A septated germ-tube zone still containing som e remnantsof vacuolated cytoplasm. Some round-shaped, dim nuclei are still evident (solid arrowhead), h Cytoplasm remnants between septa containingbrightly fluorescent nuclei, e and d In some germ tubes the presence of brightly fluorescent chromatin debris squeezed between close cleansepta is revealed . Bars: 10 ~xm( F i g. 5 e) . S m a l l , i s o l a t e d p i e c e s o f f l u o r e s c e n t m a t e -r i a l w e r e a l s o p r e s e n t .T h e c l e a n - s e p t a g e r m - t u b e z o n e i l l u s tr a t e d i n F i g s . 4a n d 5 d e x t e n d e d s e v e r a l m i l l i m e t e r s e n d i n g i n ar e g i o n i n w h i c h c y t o p l a s m r e a p p e a r e d d i s p l a y in g am o r e g r a n u l a r t e x t u r e ( F i g s . 4 a n d 5 f , l e f t) a n d c o n -t a i n i n g n u c l e i w h i c h a p p e a r e d r o u n d i s h a n d d i m m e r

t h a n t h o s e d e s c r i b e d a b o v e ( F i g . 5 f, r i g h t) . T h e s e f e a -t u re s w e r e m a i n t a i n e d f o r s e v e r a l h u n d r e d s o fm i c r o m e t e r s u p t o a s ho r t g e r m - t u b e c l e a n - s e p t a z o n e( F i g s . 4 a n d 5 g , l e f t ) , w h i c h w a s f o l l o w e d b y a n o t h e rg e r m - t u b e a r e a c o n t a i n i n g i r r e g u l a r l y s h a p e d , b r i g h tnuc l e i (F i g . 4 ; de l i mi t ed i n F i g . 5 g , h , r i gh t ) .

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B. B ago et al.: In viv o studies on nuclei of axenic Gigaspora roseaA s s h o w n in F ig s . 4 a n d 5 h - j , t h e c y to p l a s m ic d is -c o n t i n u i t i e s d e s c r i b e d a b o v e w e r e p r e s e n t a l l a l o n gth e r e s t o f th e s p e c im e n B g e r m tu b e : g r a n u l a r a n d /o rv a c u o l a t e d c y to p l a s mic z o n e s , c o n t a in in g b r i g h t l yf l u o r e s c e n t n u c l e i ( e i t h e r r o u n d - o v a l o r i r r e g u l a r l ys h a p e d ) , a l t e r n a t e d w i th z o n e s s h o w in g l e s s g r a n u l a rc y to p l a s m a n d c o n t a in in g r o u n d , d im n u c l e i . T h e tw ot y p e s o f c y t o p l a s m i c z o n e s w e r e w e l l d e l i m i t e d b ys e p t a , w h ic h s e e m e d t o s e p a ra t e d i f f e re n t c y to p l a s m ics t a t e s . D im n u c l e i w e r e mo r e d i f f i c u l t t o d i s t i n g u i s ha s o b s e r v a t i o n s w e r e c a r r i e d o u t i n g e r m- tu b e a r e a sc lo s e r t o t h e s p o r e ( c o m p a r e F ig . 5 h w i th i a n d j ).S p e c im e n C ( F ig . 6 ) c o n s i s t e d o f a n e n t i r e ly s e p t a t e dG. rosea g e r m tu b e . T r a n s mi t t e d - l i g h t ima g e s r e -v e a l e d t h a t mo s t o f t h e h y p h a l l e n g th o f th i s s p e c im e nd i s p l a y e d c l e a n s e p t a ( F ig . 6 a - d , l e f t ) a n d w a sd e v o id o f n u c l e i ( F ig . 6 a - d , r ig h t ). W h e r e r e mn a n t so f h i g h ly v a c u o l a t e d c y t o p l a s m w e r e f o u n d , s o m er o u n d i s h , d im n u c l e i w e r e n o t e d ( F ig . 6 a ) . B r ig h t l yf l u o r e s c e n t c h r o ma t in ma s s e s w e r e a l s o n o t e d c lu s -t e r e d b e tw e e n c lo s e s e p t a . I n s o me c a s e s t h e y w e r eround, nuc le i - l ike s t ruc tures (F ig . 6 b) , in o the r s theya p p e a r e d a s c o mp r e s s e d n u c l e a r d e b r i s ( F ig . 6 c , d ) .

i s cus s i onT h e m u l t in u c l e a te d a n d c o e n o c y t i c n at u re o f A M f u n -g i, a n d t h e a l m o s t c o m p l e t e l a c k o f k n o w l e d g e c o n -c e r n in g t h e i r g e n e t i c s a n d n u c l e a r c y c l e h a v e g r e a t l yh a m p e r e d p r o g r e ss o n t h e k n o w l e d g e o f t h e b i o l o g yo f t h e s e f u n g i , a n d o f a r b u s c u l a r m y c o r r h i z a l p l a n t s .A l th o u g h i n t e r e s t i n g d a t a c o n c e r n in g mo r p h o lo g y ,n u mb e r , d i s t ri b u t i o n , a n d p lo id y l e v e l o f A M f u n g a ln u c l e i h a v e b e e n r e p o r t e d , t h e f a c t t h a t a l l t h e s e s t u d -i e s w e r e c a r r ie d o u t o n f i x e d s p e c im e n s d id n o t a l l o wf o r a n i n v e s t i g a t io n o f t h e n u c l e a r b e h a v io r a l o n gh y p h a e a n d t h e f i n a l f a t e o f n u c l e i .S ma l l f u n g a l n u c l e i a r e mo r e d i f f i c u l t t o v i s u a l i z ew i th l i g h t mic r o s c o p y t h a n t h o s e o f o th e r o r g a n i s ms( H e a th 1 9 8 7 ) . I n t h i s r e s p e c t f l u o r e s c e n c e mic r o s c o p yp r o v e d mo r e u s e f u l ( B u t t e t al . 1 9 8 9 ) . A f u r t h e r s t e pin c y to lo g i c a l a n d n u c l e a r r e s e a r c h w a s a c c o mp l i s h e dw i t h t he d e v e l o p m e n t o f C L S M , w h i c h a l l o w e d o p t i -c a l s e c t i o n in g a n d 3 - a n d 4 - D r e c o n s t r u c t i o n o fima g e s , w i th s i g n i f i c a n t l y b e t t e r r e s o lu t i o n a n d c o n -t r a s t ( C z y mme k e t a l . 1 9 9 4 , P a w le y 1 9 9 5 ) . H o w e v e r ,t h e i n h e r e n t l imi t a ti o n o f f l u o r e s c e n c e mic r o s c o p y( b o th c o n v e n t i o n a l o r C L S M) i s t h a t e i t h e r t h e l a b e l -i n g a n d /o r o b s e r v a t i o n t e c h n iq u e c o u ld p e r t u r b t h ev i a b i l i t y o f t h e c e l l ( A r n d t - J o v in a n d J o v in 1 9 8 9 ) .Mu l t i p h o to n mic r o s c o p y c a n l imi t t h e p e r t u r b a t i o n

11c a u s e d b y o b s e r v a t i o n a n d p r o v i d e s u n i q u e a d v a n -t a g e s i n t h e s t u d y o f l i v in g c e l l s ( W i l l ia ms e t a l.1 9 9 4 ) . A mo n g s t t h e d i f f e r e n t D N A - s p e c i f i c p r o b e s ,D A P I h a s b e e n p o in t e d o u t a s t h e b e s t c h o i c e f o rs t u d y in g D N A - c o n t a in in g o r g a n e l l e s ( H e a th 1 9 8 7 ) ,a n d e x t e n s iv e ly u s e d i n n u c l e a r s t u d i e s o n d i f f e r e n ts p e c ime n s ( K a p u s c in s k i 1 9 9 5 ) i n c lu d in g A M f u n g i(Co oke e t a l. 1987; B ian c io t to and B onfa nte 1992,1 9 9 3 ). T h i s f l u o r o c h r o m e p r e s e n t s a v e r y h ig h a f f i n i -t y f o r d o u b l e - s t r a n d e d D N A , a t t a c h in g b y e l e c t ro s t a t -i c , n o n c o v a l e n t b o n d s t o t h e min o r g r o v e o f A - T r i c hs e q u e n c e s ( K a p u s c in s k i 1 9 9 5 ) . It is a h ig h ly s e n s i t i v e( 4 - 8 t ime s h ig h e r t h a n mi th r a my c in ; C o l e m a n e t a l.1 9 8 1 ) , s ma l l mo le c u l e ( m . w . 3 5 0 ) w h ic h i s r e a d i l ytaken u p by l iv ing ce l l s (He a th 1987, Bu t t e t a l.1989) . Be ing re la t ive ly r e s i s tan t to f ad ing (But t e t a l .1 9 8 9 ) , i t a l l o w s l o n g e r c o n t i n u e d o b s e r v a t i o n o f t h es p e c ime n , a n d , a p p a r e n t ly , d o e s n o t a f f e c t c el l v i a b i l-i t y w h e n u s e d a t l o w c o n c e n t r a t i o n s ( C o le ma n a n dG o f f 1 9 8 0 , T o d a e t a l. 1 9 8 1 ) , a s w e h a v e a l s o n o t e d i no u r p r e l imin a r y v i a b i l i t y a s s a y s .T h e u s e o f 2 P M a n d D A P I o n a x e n i c c u l t u r e s o f G .rosea h a s a l l o w e d u s t o c a r r y o u t f o r t h e f i r s t t ime i nv i v o m i c r o s c o p y s t u d i e s o n A M f u n g i , a n d t h u s t oo b t a in n e w d a t a o n t h e o r g a n i z a t i o n a n d d i s t r i b u ti o n ,b e h a v io r a n d f a te o f n u c l e i d u r in g t h e n o n s y m b io t i cp h a s e o f t h is A M f u n g u s .

Organization and distribution of nucleialong AM fung al germ tubesI n ma tu r e , n o n s e p t a t e d a x e n i c a l l y c u l t u r e d G. roseag e r m tu b e s , r o u n d o r o v a l s h a p e d n u c l e i w e r e a lmo s to mn ip r e s e n t a l o n g t h e h y p h a e . B i a n c io t t o a n d B o n -f a n t e ( 1 9 9 3 ) d e s c r i b e d r o u n d n u c l e i a s u s u a l l y l o c a t e din t h e c e n tr a l p a r t o f h y h a e , w h e r e a s e lo n g a t e d n u c l e ia p p e a r e d mo s t l y n e x t t o t h e c e l l w a l l . O u r i n v iv o2 P M o b s e r v a t i o n s g iv e s a s t e p f o r w a r d , r e v e a l i n g t h a to v a l n u c l e i a r e m o t i o n l e s s a n d r e m a i n s o m e h o w

a n c h o r e d i n l a t er a l p o s i t i o n s o f t h e g e r m tu b e ,w h e r e a s c e n t r a l l y l o c a t e d , r o u n d n u c l e i mo v e a lo n gw i th t h e c y to p l a s m ic s t r e a min g .P r e l imin a r y g e n e t i c e v id e n c e h a s s u g g e s t e d t h e e x i s -t e n c e o f d i f f e re n t n u c l e a r p o p u l a t i o n s w i th in s i n g l eA M f u n g a l s p o r e s ( S a n d e r s a n d W ie mk e n 1 9 9 7 ) . T h ef i r s t d a y a f t e r g e r min a t i o n , s o me 8 0 0 n u c l e i mig r a t eo u t o f t h e s p o r e t o t h e g r o w in g g e r m tu b e ( B 6 c a r d a n dP f e f f e r 1 9 9 3 ) . O n e c o u ld , t h u s , e n v i s a g e d i f f e r e n ts p e c i a l i z e d n u c l e i mo v in g a lo n g t h e e me r g in g g e r mtu b e ( r o u n d , mig r a t i n g n u c l e i ) t o s p e c i f i c l o c a t i o n sa c c o r d in g t o a p r e p r o g r a mme d f u n g a l g r o w th s c h e d -

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12u le , t h e n b e c o min g a n c h o r e d ( o v a l , mo t io n l e s snuc le i ) and express ing , lead ing (and , eventua l ly , d i f -f e ren t ia t ing) the ge rm- tube a rea under the i r cont ro l .T h i s w o u ld me a n t h a t d i f f e r e n t f u n g a l c o mp a r tme n t s( c e l ls ) w i t h in c o e n o c y t i c A M f u n g a l g e r m t u b e sw o u ld i n d e e d e x i s t , e v e n i n t h e a b s e n c e o f p h y s i c a lb a r r i e r s ( s e p t a ) . F u r th e r c y to lo g i c a l a n d mo le c u l a rr e s e a r c h i s n e e d e d t o v e r i f y s u c h h y p o th e s i s .A s i n o th e r f u n g i , t h e c o r r e c t d i s t r i b u t i o n o f n u c l e ia lo n g G. rosea h y p h a e w o u l d p r o b a b l y r e q u i r e t h ee x i s te n c e o f m o l e c u l a r m o t o r s t o p r o d u c e t h e c o r -r e c t n u c l e a r mo v e m e n t s , a n d s e n s o r s t o mo n i to r t h ea c tu a l p o s i t i o n o f n u c l e i i n t h e h y p h a . T h e f u n g a lc y to s k e l e to n ( c o mp r i s i n g mic r o tu b u l e s a n d F - a c t i n )s e e ms t o b e i n t ima t e ly i n v o lv e d i n t h e s e p r o c e s s e s( H e a th 1 9 9 4 ) . I n t e r e s t i n g ly , A s t r 6 m e t a l . ( 1 9 9 4 )o b s e r v e d t h a t mic r o tu b u l e s o f a x e n i c a l l y c u l t u r e dGIomus mosseae ( a n o t h e r A M f u n g u s ) w e r e l o c a t e db o th i n t h e c o r t i c a l a n d c e n t r a l p a r t s o f g e r m tu b e s ,w i th n u c l e i p o s i t i o n e d a lo n g t h e mic r o tu b u l e t r a c k s .T h e s e a u th o r s s u g g e s t e d t h a t n u c l e a r d i s t r i b u t i o n i nA M f u n g a l h y p h a e c o u l d b e a m i c r o t u b u l e -d e p e n d e n tp r o c e s s. O u r i n v iv o , 2 P M o b s e r v a ti o n s o n m o v e m e n ta n d p o s i ti o n in g o f G. margarita n u c l e i a l o n g t h e g e r mtu b e s s e e m to f u rt h e r s u p p o r t s u c h h y p o th e s i s .

Nuclear behavior following fungal growth arrestF l u o r e s c e n c e m i c r o s c o p y , C L S M a n d 2 P M o b s e r v a -t i o n s r e v e a l e d t h a t s o m e r e s tr i c t e d z o n e s o f ma tu r eg e r m tu b e s o f a x e n i c a l ly c u l t u r e d G. rosea a s w e l l a se x t e n s iv e h y p h a l z o n e s o f s p e c ime n s b e g in n in g t os e p t a te , s h o w e d c lu s t e rs o f b ri g h t l y D A P I - s t a in e dchromat in qui te of ten in i r r egula r and/or s t r and- l ikes h a p es . B o t h , t h e h i g h s p e c i f i c it y o f D A P I f o r D N Aa n d t h e s i z e o f t h e s t a i n e d i r r e g u l ar l y s h a p e dorgane l le s , ind ica te tha t these objec ts a re , in f ac t ,d e f o r me d n u c l e i . A d d i t i o n a l c o lo c a l i z a t i o n e x p e r i -me n t s u t i l i z i n g b o th D A P I a n d t h e mi to c h o n d r i a - s p e -c i f i c s t a i n M y t o T r a c k e r G r e e n F M ( M o l e c u l a rP r o b e s , E u g e n e , O r e g . , U . S . A . ) r u l e d o u t t h e p o s s ib i l -i t y t h a t t h e s e s t r u c tu r e s a r e o th e r D N A - c o n t a in in gorgane l le s ( i . e . , mi tochondr ia ) r a the r than nuc le i (da tan o t s h o w n ) . H y p h a l c l u s t e r s c o n t a in in g d i s t o r t e dn u c l e i u s u a l l y e x h ib i t e d a h ig h ly v a c u o l a t e d c y to -p l a s m, a n d w e r e d e l imi t e d b y s e p t a w h ic h i s o l a t e dth e m f r o m th e re s t o f t h e h y p h a . N e i t h e r c y to p l a s mics t r e a m i n g n o r n u c l e a r m o v e m e n t w e r e o b s e r v e d i nth e s e z o n e s . C o n s id e r e d t o g e th e r , t h e s e f e a tu r e s s u g -g e s t t h a t i n t h e s e c lu s t e r e d a r e a s a u to ly t i c p r o c e s s e sw e r e t a k in g p l a c e o w in g t o t h e l a c k o f a s y mb io t i c

B. B ago et al.: In viv o studies on nuclei of axenicGigaspora roseas w i t c h o n s i g n a l o r to a s u b s e q u e n t s i g n a l f o r f u n g a l

g r o w th a r r e s t, t h e f u n g u s n o t h a v in g f o u n d a h o s t r o o tt o c o lo n i z e . T h i s h y p o th e s i s i s b a s e d o n t h e f o l l o w in go b s e r v a t i o n s : ( i ) s u c h c lu s t e r s w e r e n e v e r n o t i c e d i ny o u n g , a c t i v e ly - g r o w in g g e r m tu b e s ; ( i i ) a b s e n c e o fc y to p l a s mic s t r e a min g a n d h ig h v a c u o l a t i o n a r e f e a -t u r e s c o mmo n ly f o u n d i n c e l l u l a r a u to ly t i c p r o c e s s e s( e .g . , p r o g r a mm e d c e l l d e a th ; G r o o v e r e t a l. 1 9 9 7 ) ;( i i i ) mo s t n u c l e i s h o w e d i r r e g u l a r s h a p e s i n t h e s ez o n e s , s o me t ime s a p p e a r in g l o b e d , s o me t ime s a sc h r o ma t in s t r an d s o r ma s s e s ( s imi l a rl y f e a tu r e dn u c l e i h a v e b e e n d e s c r i b e d i n c e l l s u n d e r g o in g p r o -g r a mme d c e l l d e a th / a p o p to t i c p r o c e s s e s ; K a t s u h a r aa n d K a w a s a k i 1 9 9 6 , G r o o v e r e t al . 1 9 9 7 ) ; a n d ( iv ) th eb r ig h t e r f l u o r e s c e n c e d i s p l a y e d b y n u c l e i a n d i r r e g u -l a r l y s h a p e d n u c l e i i n t h e s e z o n e s . T w o p o s s ib l ee x p l a n a t i o n s f o r t h e i n c r e a s e d f l u o r e s c e n c e a r e p l a s -ma me mb r a n e d a ma g e a t t h e c lu s t e r e d a r e a s , w h ic ha l l o w a n e a s i e r f l o w o f t h e d y e i n to t h e h y p h a l c y to -p l a s m, o r i n c r e a s e d D A P I f l u o r e s c e n c e d u e t oa u to ly t i c p r o c e s s e s . I n c r e a s e d D A P I f l u o r e s c e n c e h a sb e e n r e p o r t e d i n n u c l e i o f a n ima l c e l l s t r e a t e d w i tha g e n t s k n o w n to i n d u c e a p o p to s i s ( Mi l l e r e t a l. 1 9 9 7 ) .T h i s i s a p p a r e n t l y d u e t o th e b r e a k d o w n o f t h e s u p e r -c o i l i n g o r g a n i z a ti o n o f c h r o ma t in , w h ic h w o u ld r e n -d e r t h e D N A m o l e c u l e m i n o r g r o o v e m o r e a c c e s s ib l eto D A P I . S imi l a r l y , an i n c r e a s e in D A P I f l u o r e s c e n c eo f a p o p t o t ic t o b a c c o c e l ls h a s b e e n r e m a r k e d b y o n eo f u s ( H . C h a m b e r l a n d p e r s . o b s . ).O u r r e s u l t s s u g g e s t t h a t , i f t h e r e i s n o h o s t r o o t t o c o l -onize , G. rosea u n d e r g o e s a u to ly t i c p r o c e s s e s s imu l -t a n e o u s ly i n d i f f e r e n t g e r m- tu b e z o n e s . S u c h a u to ly t -i c e v e n t s a r e c h a r a c t e r i z e d b y i n c r e a s e d c y to p l a s micv a c u o l a t i o n , imm e d ia t e c e s s a t i o n o f c y to p l a s m ics t r ea min g , n u c l e a r d i s r u p t i o n , a n d a r a p id s e p t a t i o n o ft h e a f f e c t e d z o n e i n o r d e r t o l imi t d a m a g e s . T h e p r e s -e n c e o f r a n d o m a u t o ly s i s h a s a l re a d y b e e n s h o w n i no th e r f i la me n to u s f u n g i ( F e n c l 1 9 7 8 ) , a n d w a s a t t r ib -u t e d t o r a n d o m a n d l o c a l i z e d a g e in g e v e n t s . T r in c ia n d R ig h e l a to ( 1 9 7 0 ) c o n f i r me d t h a t i n f i l a me n to u sf u n g i a u to ly s i s i s n o t a s y n c h r o n o u s p r o c e s s t h r o u g h -o u t t h e e n t i r e h y p h a , b u t o n ly i n i n d iv id u a l c o mp a r t -m e n t s . H o w e v e r , w i t h i n a l y s e d c o m p a r t m e n t t h eb r e a k d o w n o f o r g a n e l l e s i s s y n c h r o n o u s . T h e r e s u l t sp r o v id e d h e r e c o n s t i t u t e t h e f i r s t e v id e n c e o f a s imi l a rb e h a v io r i n A M f u n g i , w h e r e g r o w th a r r e s t w a s s o f art h o u g h t t o o c c u r t h r o u g h c y to p l a s mic r e t r a c t i o n a n ds e p t a fo r ma t io n s t a rt in g f r o m th e g e r m- tu b e a p e x a n dto w a r d s t h e s p o r e ( e . g . , Mo s s e , 1 9 6 2 , B o n f a n t e a n dP e r o t t o 1 9 9 5 ) .W h e t h e r t h e s e r a n d o m a u t o l y ti c e v e n ts u n d e r g o n e b y

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B. Bag o e t a l. : I n v ivo s tud i es on nuc l e i o f axen ic Gigaspora rosea 13a s y m b i o t i c G ro se a g e r m t u b e s a r e p r o g r a m m e d( s ta r t in g a s a c o n se q u e n c e o f t h e l a c k o f t h e sy m b io t -i c sw i t c h o n s ig n a l) o r n o t , r e ma in s t o b e e lu c id a t -e d . A s d i s c u s se d b y G r o o v e r e t a l. ( 1 9 9 7 ) t h e k e ya sp e c t o f p r o g r a m me d c e l l d e a th i s t h at t h e d y in g c e l li s i t se l f pa r t ic ipa t ing ac t ive ly in i ts ce l lu la r d isassem -b ly b y a n o r d e r e d a n d c o n c e r t e d s e t o f a c t i o n s . P r o -g r a mme d c e l l d e a th h a s b e e n a l r e a d y f o u n d th r o u g h -out the an ima l and p lan t k ingdoms (El l i s e t a l . 1991,M ar t in e t a l. 1994 , Gro ov er e t a l. 1997, Kos s lak e t a l.1 9 9 7 , L e g r a n d 1 9 9 7 , N o o d e n e t a l. 1 9 9 7 ), a n d mo r er e c e n t ly su g g e s t e d t o o c c u r a l so i n f u n g i ( U ma r a n dV a n g r i e n sv e n 1 9 9 7 , J a c o b so n e t a l . 1 9 9 8 ) . I t i st h o u g h t t o b e a s e l e c ti v e me c h a n i sm th a t a l lo w s c e l l st o s u r v i v e o n l y w h e n a n d w h e r e t h e y a r e n e e d e d(Albe r ts e t a l . 1994 , Legrand 1997) . More re sea rch i sr e q u i r e d t o c l a r i f y it s p o s s ib l e o c c u r r e n c e i n A M f u n -gi.

F a te o f n u c l e i r e l e a se d a f t e r sp o re g e rm in a t io nI n c o m p l e t e l y s e p t a t e d h y p h a e s o m e r e m n a n t s o fc h r o m a t i n a p p e a r e d c o m p r e s s e d b e t w e e n c l o s e l yne ighbour ing sep ta . This sugges ts tha t a t leas t a pa r to f t h e n u c le i c o m i n g o u t o f t h e A M f u n g a l s p o r e a ft e rg e r min a t i o n d o n o t r e e n t e r i t ( a s i t w a s c o mmo n lyth o u g h t ) , b u t r e ma in i n t h e s e p t a t e d h y p h a a n d e v e n -tu a l l y d e g r a d e . T h u s , a n d s in c e A M f u n g a l sp o r e sp r e se n t t h e c a p a c i t y o f mu l t i p l e r e - g e r min a t i o n s( H e p p e r 1 9 8 4 , B a g o 1 9 9 0 ) o n e c o u ld t h in k t h a t t h en u m b e r o f n u c l e i c o n t a in e d i n a s in g l e sp o r e w o u ldd e c r e a se w i th i n c r e a se d n u mb e r o f r e - g e r min a t i o n s .H o w e v e r , B 6 c a r d a n d P f e f f e r ( 1 9 9 3 ) f o u n d th a ta l t h o u g h th e n u m b e r o f n u c l e i i n t h e sp o r e d e c l in e dma r k e d ly ( b y 8 0 0 ) o n e d a y a f t e r g e r min a t i o n , i tr e c o v e r e d s lo w ly l a t e r , p r o b a b ly d u e t o mi to t i ce v e n t s , a n d a lmo s t r e a c h e d i t s o r i g in a l v a lu e . T h u s ,t h e r e c o v e r y o f a ll n u c l e i r e l e a se d e a r l i e r s e e ms n o t t ob e n e c e s sa r y f o r th e n e w ly d o r ma n t sp o r e . T h e se f i n d -in g s , h o w e v e r , r a i s e i n t e r e s t i n g q u e s t i o n s c o n c e r n in gth e t y p e o f n u c l e i t h a t a r e l o s t f o l l o w in g sp b r e g e r mi -n a t i o n a n d a sy mb io t i c f u n g a l g r o w th a r r e s t , f o rin s t a n c e , d o t h e y a l l b e lo n g t o s imi l a r p o p u la t i o n s( e .g . , t h e g e r min a t i o n a n d fi n d in g o f a h o s t r o o tn u c l e a r p o p u la t i o n s ) ? T h i s w o u ld r e su l t i n c e r t a inn u c l e a r p o p u la t i o n s w i th in a sp o r e d e c r e a s in g i n n u m -b e r w i th r e sp e c t t o o th e r s . T h e d e v e lo p me n t o f a d e -q u a t e mo le c u l a r b io lo g y t o o l s a l l o w in g u s t o a d d r e s sth i s a n d o th e r f u n d a me n ta l q u e s t i o n s i s i n d e e drequi red .I t i s w e l l k n o w n th a t , i n a b se n c e o f so i l d i s t u r b a n c e ,

t h e A M f u n g a l my c e l iu m r e t a in s i t s c o lo n i z a t i o np o te n t i a l, e v e n i f h y p h a e a r e o ld a n d /o r e n v i r o n me n -ta l condi t ions a re adverse ( Jaspe r e t a l . 1989) . Warne ra n d Mo sse ( 1 9 8 0 ) r e p o r t e d t h a t so me h y p h a e i n t h eso i l w e r e s t il l c a p a b l e o f c o lo n i z in g p l a n t s e v e n i fd e t a c h e d o f t h e o r ig in a l i n o c u lu m . I n t e r e s ti n g ly , o u r2 P M o b se r v a t i o n s r e v e a l e d t h a t d u r in g t h e g e r m- tu b ese p t a t i o n p r o c e s s , a n d e v e n i n f u l l y s e p t a t e d h y p h a e ,c y to p l a smic c lu s t e r s c o n t a in in g n o r ma l - l o o k in gn u c l e i w e r e p r e se n t . I t i s t e mp t in g t o a s so c i a t e su c h

f o r g o t t e n n u c l e i w i th t h e ma in t e n a n c e o f t h e i n f e c -t i v i t y p o t e n t i a l o f A M f u n g a l h y p h a e u n d e r n a tu r a lc o n d i t i o n s . H o p e f u l l y f o r th c o m in g r e se a r c h w i l l c la r -i fy th is po in t .cknowledgements

T he au tho r s g r a t e f u l ly acknowledge the t echn ica l suppor t o f Ju l i eS a m s o n ( C R B F , U . L a v a l ) a n d K e n n e t h O r n d o r f f ( D a r m o u t h M e d -i ca l Schoo l , Hanover , N .H. , U .S .A. ) . Spec i a l t hanks a r e due t oF r a n q o i s L a r o c h e l l e , M a u r i c e L a l o n d e a n d A l i c e R o y ( C R B F , U .L ava l ) . B . B . a l so du ly acknowledges t he f acu l ty and co l l eagues o fthe 1s t Cour se on 3 - D M icr o scopy o f L iv ing Ce l l s ( Vancouver ,1996) , and the f i nanc i a l a ss i s t ance p r ov ided by Kar l Z e i ss C anada .T h i s wor k has been suppor t ed by two pos tdoc to r a l f e l l owsh ips t o B .B. ( Di r ecc i6n G ener a l de I nves t i ga c i6n Cien t f f i ca y T dcn ica , Spa in ,and M in i s t6 r e de l l~duca t ion , Qu6bec) , a s w e l l a s by NSE R Cr esea r ch g r an t s t o Y . P . and J . G . L . and by NI H/NCCR f und ing o ft h e D e v e l o p m e n t a l R e s o u r c e f o r B i o p h y s i c a l I m a g i n g a n d O p t o e l e c -tronics at Cornelt Unive rsity (W. Z., R. M. W., and W . W. W .).

e f e r e n c e sA l b e r t s B , B r a y D , L e w i s J , R a f t M , R o b e r t s K , W a t s o n J D ( 1 9 9 4 )

M ole cu la r b io logy o f t he ce ll , 3 r d edn . Gar l and Pub l i sh ing , NewY o r k

A r n d t - J o v i n D J , J o v i n T M ( 1 9 8 9 ) F l u o r e sc e n c e l a b e l i n g a n dm i c r o s c o p y o f D N A . M e t h o d s C e l l B i o l 3 0 : 4 1 7 - 4 4 8

2~st r6m H, Giovanne t t i M , Raud askosk i M ( 1994) Cy toske l e t a l com-ponen t s i n t he a r buscu la r mycor r h i za l f ungus Glomus mosseaeM o l P l a n t M i c r o b e I n t e r a c t 7 : 3 0 9 - 3 1 2

Azc6 n- Ag u i l a r C , Bago B ( 1994) Phys io log ica l cha r ac t e r is t i c s o f t heh o s t p l a n t p r o m o t i n g a n u n d i s t u r b e d f u n c t i o n i n g o f t h e m y c o r -r h i za l symbios i s . I n : Gian inazz i S , Sch t i epp H ( eds) I mpac t o fa r buscu la r mycor r h i zas on sus t a inab le ag r i cu l tu r e and na tu r a lecosys t ems . Bi r kh~ iuser , Base l , pp 47 - 60 .

- B a r e a J M ( 1 9 9 5 ) S a p r o p h it i c g r o w t h o f A M F . I n : V a r m a A ,Hock B ( eds) M ycor r h i za : s t r uc tu r e , f unc t ion , mo lecu la r b io lo -gy , and b io t echno logy . Sp r inger , Ber l i n He ide lbe r g New Yor kT o k y o , p p 3 9 1 - 4 0 7

Bago B ( 1990) E f ec to de d i s t i n tos compues tos azu f r ados sob r e e lc r e c i m i e n to i n d e p e n d i e n t e d e l h o n g o f o r m a d o r d e m i c o r ri z a s v aGlomus mosseae M a s t e r t h e s i s , U n i v e r s i d a d d e G r a n a d a ,G r a n a d a , S p a i n

- Vie r he i l i g H , P i ch6 Y, Azc 6n- A gu i l a r C (1996) Ni t r a t e dep le t i ona n d p H c h a n g e s i n d u c e d b y t h e e x t r a r a d i c a l m y c e l i u m o f t h ea r b u s c u l a r m y c o r r h i z a l f u n g u s Glomus intraradices g r o w n i nm o n o x e n i c c u l t u r e . N e w P h y t o l 1 3 3 : 2 7 3 - 2 8 0

8/12/2019 Bago et al_1998

14/15

14 B. Bag o et al .: In vivo studies on nucle i of axenic Gigaspora roseaBago B , Ben t ivenga SP , B renac V, Dodd JC , P ich6 Y, S imon L

(1998) Molecu lar analysis of Gigaspora (Glomales , Giga-sporaceae). New Phytol 139 (in press)

Balestr ini R, Bianciotto V, Bonfante-Fasolo P (1992) Nuclear archi-tec tu re and DNA loca t ion in two VAM fung i . Mycorrh iza 1 :105-112

Barea JM, Jeffries P (1995) Arbuscnlar mycorrhizas in sustainablesoil plant systems. In: Varma A, Hock B (eds) Mycorrhiza:structure, function, m olecula r biology, and biotechnolog y.Spr inger, Ber l in He ide lbe rg New York Tokyo , pp 5 21-560

B6card G , Pfeffer PE (1993) Status o f nuclear division in arbuscularmycorrh iza l fung i du r ing in v i t ro deve lopment . P ro top lasma1 7 4 : 6 2 - 6 8

- Pich6 Y (1989) Fungal growth stimulation by CO2 and root exu-dates in vesicular-arbu scular mycorrhiz al symbiosis. Appl Envi-r o n M i c r o b i o 1 5 5 : 2 3 2 0 -2 3 2 5

Bianc io t to V, Bonfan te P (1992) Quan t i f ica t ion o f the nuc lea r DNAcontent of two arbuscular myc orrhizal fungi. M yco l Res 96:1071-1076

- - (1993) Evide nce of DN A replication in an arbuscular mycor -rhizal fungus in the absence of the host plant. Proto plasma 176:100-105

Bonfan te P , B ianc io t to V (1995) P resymbio t ic ve rsus symbio t icphase in arbuscular e ndom ycorrhiza l fungi: morpholo gy andcytology. In: Varma A, Hock B (eds) Mycorrhiza: s tructure,func t ion , molecu la r b io logy , and b io techno logy . Spr inger ,Ber l in He ide lbe rg New York Tokyo , pp 229-247

- Perotto S (1995) Strategies of arbuscular myc orrhizal fungiwhen in fec t ing hos t p lan ts , New Ph y to l 130 :3 -21

Bonfan te -Faso lo P , Ber ta G, Fuscon i A (1987) D is t r ibu t ion o f nuc le iin a VA M dur ing i t s symbio t ic phase. T rans Br M yco l Soc 88 :2 6 3 - 2 6 6

Burggraa f AJP , Ber inger JE (1989) Absence o f nuc lea r DNA syn-thesis in vesicular-arbuscular mycorrhizal fungi during in vitrodeve lopment . New Phy to l 111 : 25 -33

But t TM, Hoch HC, S tap les RC, S t Leger RJ (1989) Use o f f luo -rochromes in the study of fungal cytology and differentiation.E x p M y c o l t 3 : 3 0 3 - 3 2 0

Coleman AW, Goff LJ (1980) App l ica t ions o f f luo rochromes topo l len b io logy I : mi th ramycin and 4 ,6 -d iamid ino-2 -pheny l in -dole (DA PI) as vital s tains and fo r quantitation of nuclear DN A.S ta in Techno l 60 :145-154Mag uire MJ, Colema n JR (1981) Mithra my cin and 4 ,6-diamidi-no-2 -pheny l indo le (DAPI) : DNA s ta in ing fo r f luo rescencemicrospectrophotometric measurements in nuclei , plastids andv i rus pa rt ic les . J His tochem 29 :959-9 68

Cooke JC , Gem ma JN, Koske R E (1987) Observa t ions o f nuc le i inves icu la r-a rbuscu lar mycorrh iza l fung i. M yco log ia 79 : 33 1- 3 3 3

Czymmek KJ , Whal lon JH, Klomparens KL (1994) Confoca lmic roscopy in myco log ica l re sea rch . Exp M yco l 18 : 275 -293

Denk W , S t r ick le r JH, Webb WW (1990) Tw o-pho ton la se r scann ingf luorescence mic roscopy . Sc ience 248 :73-7 6P is ton DN, Webb W W (1995) Two -pho ton molecu la r exc ita t ionin laser scanning fluorescence microscopy. In: Pawley JB (ed)Handbook o f b io log ica l confoca l mic roscopy . P lenum, NewYork , pp 445-458

El l i s R , Yuan J , Horv i tz R (1991) Mechan isms and func t ions o f ce l ldeath . Annu Rev Ce l l B io l 7 :66 3-69 8

Fenc l Z (1978) Ce ll ageing and autolysis . In: Sm ith JE, Berry DR(eds) The f i lamen tous fung i , vo l 3 , deve lopmenta l myco logy .Ha ls ted P ress, N ew Y ork , pp 389-405

Gerdemann JW (1955) R e la t ion o f a la rge so i l -borne spore to phy-comyce tous myco rrh iza l in fec t ions. Myco log ia 47 :619- Nico lson JH (1963) Spores o f mycorrh iza l Endogone speciesextracted from soil by wet-sieving and decanting. Trans BrM y c o l S o c 4 6 : 2 3 5 - 2 4 4

Giovanne t t i M, Sbrana C , Av io L , C i te rnes i AS , Log i C (1994)Recognition and infection process, basis for host specificity ofarbuscular my corrhizal fungi. In: Gianin azzi S, Schiiepp H (eds)Imp act of arbuscular myc orrhizas on sustainable agriculture andnatural eco systems. BirkNiuser, Basel, pp 61-7 2

Groover A, De W it t N, He ide l A, Jones A (1997) P rogram med ce l ldeath of plant tracheary elements differentiating in vitro. Proto-p lasma 196 :197-211

Heath IB (1987) Flu orescen t staining of fungal nuclei . In: Fuller MS,Jaworski A (eds) Zoosporic fungi in teaching and research.Southeastern Publishing, Greenv ille , pp 16 9-171

- (1994) The cy toske le ton in hypha l g rowth , o rgane l le movem entsand mi tos is . In : Wesse ls JGH , Meinhard t F (eds ) The M yco ta ,vol 1 , growth, differentiation and sexuality . Springer, BerlinHeide lbe rg New Yo rk Tokyo , pp 43-65

Hepper C (1983) L imi ted independen t g rowth o f a ves icu la r -a rbus-cu la r mycorrh iza l fungus in v it ro . New Phy to l 93 :5 37-5 42(1984) Isolation and culture of VA mycorrhizal (VAM) fungi.In : Powel l CL , Bagyara j D J (eds) VA mycorrh iza . CRC Press ,Boca Ra ton , pp 95-112

Jasper DA, Abbo t t LK, Robson A D (1989) Hyphae o f ves icu la r -arbuscular myc orrhizal fungus main tain infectivity in dry soil ,excep t when the so i l i s dis tu rbed . New Phy to l 11 2 :101-1 07

Jacobson DJ , Beurkens K, Klomparens K L (1998) Microscop ic andultrastructural exam ination of vege tative incompatibil i ty in par-tial diploids heterozygous at het loci in Neurospora crassa Fun-g a l G e n B i o l 2 3 : 4 5 - 5 6

Kapusc insk i J (1995) DA PI : a DN A-spec i f ic f luo rescen t p robe .B i o t e c h H i s to c h e m 7 0 : 2 2 0 - 2 3 3

Katsuhara M, Kawasaki T (1996) Salt s tress induced nuclear andDNA degrada t ion in mer is temat ic ce l l s o f ba r ley roo ts . P lan tC e l l P h y s io l 3 7 : 1 6 9 - 1 7 3

Koss lak RM, Cbamber l in MA, Pa lmer RG, Bowen BA (1997) P ro -g rammed ce l l dea th in the roo t co r tex o f soybean roo t nec ros ismutants. Plant J 11: 729-745

Legran d EK (1997) An adaptationist view of apoptosis. Q Rev Biol7 2 : 1 3 5 - 1 4 7Marsh BA B (1971) Measurem ent o f leng th in random ar rangemento f lines . J App l Eco l 8 :26 5-26 7

Martin SJ, Green DR, Cotter TG (1994) Dicing with death: dissect-ing the componen ts o f the apop tos is mach inery . T rends B iochemS c i 1 9 : 2 6 - 3 0

Meie r R , Charva t I (1992) Germina t ion o f Glomus mosseae spores:proc edu re and ultrastructural analysis. Int J Plan t Sci 153:541-549

Mil le r T , Beausang LA, Menegh in i M, L idgard G (1997) Dea th -induced changes to the nuclear matrix: the use of anti-nuclearmatrix antibodies to study agents of apoptosis . BioT echniqu es15 : 1042-1047

Mo sse B (1959) The regu lar germination of resting spores and some

8/12/2019 Bago et al_1998

15/15

B. Bago e t a l. : I n v ivo s tud i es on nuc l e i o f axen ic Gigaspora roseao b s e r v a t i o n s o n t h e g r o w t h r e q u i r e m e n t s o f a n Endogone sp.c a u s i n g v e s i c u l a r - a rb u s c u l a r m y c o rr h i z a s . T r a n s B r M y c o l S o c4 2 : 2 7 3 - 2 8 6

- ( 1 9 6 2 ) T h e e s t a b l is h m e n t o f v e s i c u l a r- a r b u s c u l a r m y c o r r h i z au n d e r a s e p t ic c o n d i t io n s . J G e n M i c r o b i o l 2 7 : 5 0 9 - 5 2 0( 1988) Som e s tud i es r e l a t ed t o i ndep ende n t g r owth o f ves i cu -l a r -a r b u s c u l a r e n d o p h y t e s . C a n J B o t 6 6 : 2 5 3 3 - 2 5 4 0N o o d e n L D , G u i a m e t J J , J o h n I ( 1 9 9 7 ) S e n e s c e n c e m e c h a n i s m s .P h y s i o l P l a n t 1 0 1 : 7 4 6 - 7 5 3

P a w l e y J B ( e d ) ( 1 9 9 5) H a n d b o o k o f b i o l o g ic a l c o n f o c a l m i c r os c o p y .P l e n u m , N e w Y o r k

S a n d e r s I R , W i e m k e n A ( 1 9 9 7 ) T h e d i v e r s i t y o f A M f u n g i a n d i t se c o l o g i ca l s i g n i f ic a n c e . I n : 4 8 t h A n n u a l M e e t i n g o f t h e A m e r i -can I ns t i t u t e o f Bio log ica l Sc i ences , Augus t 3 - 7 , 1997, Pa l a i sdes Congr ~s de M ont r da l , M on t r 6a l , Canada , p 120

S m i t h S E , R e a d D J ( 1 9 9 7 ) M y c o r r h i z a l s y m b i o s i s. A c a d e m i c P r e s s ,S a n D i e g o

Swar d R J ( 1981 a ) T he s t r uc tu r e o f t he spo r es o f Gigaspora margarita I : th e d o r m a n t s p o re . N e w P h y t o l 8 7 : 7 6 1 - 7 6 8- ( 1981b) T he s tr uc tu r e o f t he spo r es o f Gigaspora margarita II:

c h a n g e s a c c o m p a n y i n g g e r m i n a ti o n . N e w P h y t o l 8 8 : 6 6 1 - 6 6 6- ( 1981c) T he s t ruc tu r e o f t he spo r es o f Gigaspora margarita III:

g e r m - t u b e e m e r g e n c e a n d g r o w th . N e w P h y t o l 8 8 : 6 6 7 - 6 7 3T oda T , Yama moto M , Yanag id a M ( 1981) Sequ en t i a l a l t e r a t i ons i n

the nuc l ea r ch r omat in r eg ion du r ing mi tos i s o f t he f i s s ion yeas tSchizosaccharomyces pombe: v i d e o f l u o r e s c e n c e m i c r o sc o p y o fs y n c h r o n o u s g r o w i n g w i l d - ty p e a n d c o l d - s e n s it i v e c d c m u t a n t s

15by us ing a DNA - b ind ing f l uo r esce n t p r obe . J Ce l l Sc i 52 :2 7 1 - 2 8 7

T r inc i APJ , Righe l a to RC ( 1970) Changes i n cons t i t uen t s and u l t r a -s t r uc tu r e o f hypha l compar tmen t s du r ing au to lys i s o f g lucosas t a r ved Penicillum chysogenum. J G e n M i c r o b i o l 6 0 : 2 3 9 -2 4 9

U m a r M H , V a n g r i e n s v e n L J L D ( 1 9 9 7) M o l - p h o g en e t ic c e ll d e a t h i nd e v e l o p i n g p r i m o r d i a o f Agaricus bisporus. M y c o l o g i a 8 9 :2 7 4 - 2 7 7

V i e r a A , G l e n n M G ( 1 9 9 0 ) D N A c o n t e n t o f v e s ic u l a r -a r b u s c u l a rm y c o r r h i z a l fu n g a l s p o re s . M y c o l o g i a 8 2 : 2 6 3 - 2 6 7

W a r n e r A , M o s s e B ( 1 9 8 0 ) In d e p e n d e n t s p r e a d o f v e s i c u la r - a r b u s c u -l a r m y c o r r h i z a l fu n g i i n s oi l. T r a n s B r M y c o l S o c 7 4 : 4 0 7 - 4 1 0

Wa t r ud L S , He i thaus I I I J J , Jawor sk i A ( 1978) G eo t r op i sm in t hee n d o m y c o r r h i z a l f u n g u s Gigaspora margarita. M y c o l o g i a 7 0 :4 4 9 - 4 5 2

W i l l i a m s R M , P i s t o n D W , W e b b W W ( 1 9 9 4 ) T w o - p h o t o n m o l e c u -l a r exc i t a t i on p r ov ides i n t r i n s i c 3 - d imens iona l r e so lu t ion f o rl a s e r -b a s e d m i c r o s c o p y a n d m i c r o p h o t o c h e m i s tr y . F A S E B J 8 :8 0 4 - 8 1 3

X u C , W e b b W W ( 1 9 9 6 ) M e a s u r e m e n t o f t w o - p h o t o n e x c i t a t i o ncr oss sec t i ons o f mo lecu la r f l uo r ophor es wi th da t a f r om 690 to1 0 5 0 n m . J O p t S o c A m B 1 3 : 4 8 1 - 4 9 1

- Z i p f e l W , S h e a r J B , W i l l i a m s R M , W e b b W W ( 1 9 9 6 ) M u l t i p h o -ton f l uo r escence exc i t a t i on : new spec t r a l windows f o r b io log i ca ln o n - l i n e a r m i c r o s c o p y . P r o c N a t l A c a d S c i U S A 9 3 :1 0 7 6 3 - 1 0 7 6 8