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Protoplasma (1997) t97 :182-18 7 Pf lOTOPL MA9 Springer-Verlag 1997Printed in Austria

P r o t e i n s y n t h e s i s a n d G o l g i - m e d i a t e d v e s i c l e t r a f f i c i s n o t r e q u i r e d t o m a i n t a i na p o l a r is e d m e m b r a n e v o l t a g e in t h e p r e s e n c e o f a u x i nG . T h i e P ' * , A . B r i i d e r n 1, a n d A . M o r o n i 2

Pflanzenphysiologisches Institut, Un iversitiit G~Sttingen, G~Sttingen and 2 Sezione di Fisiologia e Biochim ica della Piante, U niversitadi M ilano, MilanoReceived October 28, 1996Accepted January 7, 1997

Summary. The contr ibution of protein synthesis and secretion toindol acetic acid (IAA) induced polarisation of the plasma mem branevoltage (VM) was investigated. The VMof coleoptiles from Zea mayswas measured in the presence of known inhibitors of protein- andRN A synthesis, as well as those of Golgi-mediated vesicle secretion.Inhibitors were applied under conditions at which they are known toabolish IA A stimulated H+ secretion and cell elongation effectively.Cyclohex imide (CH I), an inhibitor of protein synthesis, caused depo-lari sati on o f I.q,~iwith a half maximal concentration of approximately20 vtM. A t 100 viM CH I, k~ depo larised to a new stable voltage witha half time of 9.8 + 0.6 m in. The temporal s imilarity of CHI-induceddepolarisation and cessation of coleoptile elongation suggests thatthe induced ch ange in VM underlies inhibition of elongation. C HIevoked membrane depolarisation to a final voltage of about-100 mV irrespective of the presence or absence of auxin in theexternal m edium. Th us, CHI probably affected constitutive m em-brane transport properties independently of IAA-induced m odulationof transport proteins. C ordycepin (CO R), an inhibitor of RN A syn-thesis, had no significant effect at 40 0 ~tM on VM of IAA -treatedcells, suggesting that gene transcription for transport- or regulatoryprotein synthesis was not essential for IAA -generated polarisation ofVM. Brefeldin-A (B FA ), an inhibitor o f Golgi-med iated vesiclesecretion in maize coleoptiles, had no perceivable effect at 20 rag/1on VM of IA A-treated coleoptile cells, dem onstrating that constitu-tive or IAA-stimulated protein secretion was not essential for themechan ism underlying IAA -evoked V polarisation. Hence, IAA -stimulated and COR/BFA-depressed H+ extrusion in elongatingcoleoptiles may not be entirely mediated by auxin-enhanced ATP aseactivity.Keywords: Auxin; Maize coleoptiles; Membrane potential; Cyclo-heximide; Protein synthesis.

*Correspondenc e and repri nts: Pflanzenphysiologisches Institut,Universitfit G~Sttingen, U nte re KarspiJle 2, D -37 07 3 G6ttingen,Federal Republic of Germany.

Abbrevations: COR cordycepin; CHI cycloheximide; BFABrefeldin-A; IAA indol acetic acid.

I n t r o d u c t i o nI n h i b i t o r s o f p r o t e i n s y n t h e s i s h a v e b e e n f o u n d t oa b o l i s h a u x i n - i n d u c e d H + e f f l u x a n d c e l l e l o n g a t i o nr a p i d l y i n a v a r i e t y o f g r o w i n g c e l l s y s t e m s ( B a t e sa n d C l e l a n d 1 9 7 9, C h o a n d H o n g t 9 9 5 , H a g e r e t a l.1 9 9 1 , E d e t m a n n e t a l. 1 9 8 9 ) . F r o m t h e r a p i d e f f e c t o ft h e s e i n h i b i t o r s i t h a s b e e n c o n c l u d e d t h a t p r o t e i ns y n t h e s i s i s o f ce n t r a l i m p o r t a n c e t o t h e m e c h a n i s mo f h o r m o n e - s t i m u l a t e d c e ll e l o n g a t io n ( E d e l m a n ne t a l. 1 9 8 9 , C h o a n d H o n g 1 9 9 5 ). A s a m o d e l f o r ac a u s a l l in k b e t w e e n p r o t e i n s y n t h e s i s a n d e l o n g a t i o ni t w a s s p e c u l a t e d t h a t t h e a u x i n i n d o l e a c e t i c a c i d( I A A ) c o n t r o l s s y n t h e s i s a n d s e c r e t i o n o f a s h o rt -l i v e d p r o t e i n t o r e g u l a t e t h e a c t i v i t y o f t h e p r o t o np u m p a n d t h e r e b y H + e x t r u s i o n ( L a d o e t a l. 1 9 7 7 , C h oa n d H o n g 1 9 9 5 ). A n a l t e rn a t i v e h y p o t h e s i s e x p l a i n st h e li n k b e t w e e n p r o t e i n s y n t h e s i s a n d c o l e o p t i l ee l o n g a t i o n b y t h e a b i l it y o f a u x in t o s t i m u l a t e a r a p i dr i se i n th e c o n c e n t r a t i o n o f A T P a s e m o l e c u l e s i n t h ep l a s m a m e m b r a n e ( H a g e r e t a l. 1 9 9 1 ) . S u c h a h o r -m o n e - g e n e r a t e d r is e i n A T P a s e c o n c e n t r a t i o n a n dc o n s e q u e n t l y H + p u m p i n g m a y b e t h e m e c h a n i s mu n d e r l y i n g a c i d i f i c a t i o n o f t h e c e l l w a l l, a p r o c e s sb e l i e v e d t o b e e s s e n t i al f o r c e ll e l o n g a t i o n ( H a g e re t a l. 1 9 9 1 ). S i n c e t h e r i s e i n A T P a s e c o n c e n t r a t i o nw a s a l s o ra p i d l y r e v e r s e d b y t h e p r o t e i n s y n t h e s i si n h i b i to r c y c l o h e x i m i d e o r th e R N A s y n t he s i s

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G. T h ie l e t a t .: M a in tenan ce o f po la r i s ed mem br ane vo l t age I 83i nh ib i to r c o r dyc e p in , i t w a s a s sum e d tha t t he A TP a sep r o t e in i s sub je c t t o ra p id t u r nove r , a nd the r e f o r e t ha tgene t ransc r ip t ion (Fr ias e t a l. 19 96) , pro te in synth e-s i s a nd se c r e t i on f r om the ER to t he p l a sm a m e m -br a ne i s e s se n t i a l f o r c e l l e long a t ion ( H a ge r e t a l.t 9 9 t ) . T h e l a t t e r h y p o t h e s i s i s m a i n l y b a s e d o n t h ef ind ing tha t a ux in e nha nc e s t r a nsc r ip t ion o f t he p l a s -m a m e m b r a ne H +- A TP a se in c o l e op t i l e s ( F r ia s e t a t .1996 ) a nd tha t t he r a t e o f c e l l e longa t ion c lo se ly f o l -l ow s the t im e c ou r se o f I A A - s t im u la t e d a nd inh ib i to r -d e c r e a s e d A T P a s e c o n c e n t ra t i o n i n t h e p l a s m a m e m -b r a n e. T h e p r o p o s e d s c e n a r io w o u l d a l s o e x p la i n w h yinh ib i to r s o f i n t r a c e l lu l a r ve s i c l e t r a f f i c suc h a sB r e f e l d i n - A a n d / o r m o n e n s i n r a p i d l y i n h i b i t I A A -s t im u la t e d a c id i f i c a t i on o f t he c e l l w a l l a nd e longa -t i on o f g r o w i n g t i s s u e ( C u n n i n g h a m e a n d H a i l 1 9 8 6 ,S c h ind le r e t a l. 1994 , C ho a nd H ong 1995) . The m i s s -ing e v ide n c e in suppo r t o f t he hypo the s i s i s t ha t i t ha sno t ye t be e n show n tha t I A A a nd inh ib i to r - ge ne r a t e dc ha nge s i n A TP a se c onc e n t r a t i on c o r r e l a t e w i thA TP a se a c t iv i t y i n t he p l a sm a m e m br a ne ( N a p ie r a ndV e n i s 1995 ) . F u r the r m or e , t he p r e d i c t e d de pe nde nc yo f i n v ivo A TP a se a c t iv i t y on c on t inuous s e c r e t i onf r o m t h e E R t o t h e p l a s m a m e m b r a n e r e m a i n s t o b et e s t e d . To a dd r e s s t he se que s t ions w e m e a su r e d them e m br a ne vo l t a ge ( rv~ ) o f m a iz e c o t e op t i l e c e ll s a sa n ind i r ec t m e a s u r e o f A TP a se a c t iv i t y ( S e nn a ndG old sm i th 1988 ) . The e f f e c t s o f kno w n inh ib i to r s o fp r o t e in a nd R N A syn the s i s a nd o f i n tr a c e l lu l a r ve s i c l et r a ff i c w e r e c om p a r e d w i th t he p r e d i c t i ons o f t heh y p o t h e s e s , n a m e l y t h a t I A A s t i m u l a te s d e n o v o s y n -the s i s a nd se c r e t i on o f p r o t e in s w h ic h r e gu la t eA TP a se a c t iv i t y ( La do e t a i. 1977 , C ho a nd H ong1995) o r syn the s i s a nd inc o r po r a t ion o f a c ti ve A TP a sem o le c u le s i n to t he p l a sm a m e m b r a ne ( H a ge r e t a l.1991) .

s egmen t s ( 1 cm long) t h r eaded on a s t ee l r od and ke p t i n ae r a t eds o lu t ion ( 2 mM KC 1, unbuf f e r ed) i n t he dar k . C o leop t i l e l eng th wasob ta ined f r om the p r o j ec t ion ( 75 f o ld magn i f i ca t ion) o f a co l eop t i l ep l aced on an over head p r o j ec to r .

R e s u l t sT h e m e m b r a n e v o l t a g e ( VM ) a c r o s s t h e p l a sm a m e m -br a ne o f m a iz e c o l e op t i l e c e l l s c a n be e xp la ine d a s t hevo l t a ge a t w h ic h a n A T P a se - ge ne r a t e d ou tw a r d H i sba l a nc e d by a n inw a r d K + c u r r e n t t h r ough a K +inw ard rec t i f ie r (Rf ick e t a l. 1993, H edr ic h e t a l.1995, Thie l e t a l. 1996) . Assu mi ng tha t the IA A-induc e d po la r i s a t i on o f t he m e m br a ne i s p r e dom i -na n t ly due to a n inc r e a se i n H +- A TP a se a c t iv i t y( r e v i e w e d in N a p ie r a nd V e n i s 1995 ) , r e c o r d ing VM i sa conven ient , a lb e i t indi rec t , a ssay o f H+-32FPasea c t iv i t y . O n th i s ba s i s t he hypo the s i s t ha t I A Ainc r e a se s t he c onc e n t r a t i on o f a c t i ve H +- A TP a se s i nthe p l a sm a m e m br a ne a nd tha t t he ho r m one - induc e dr i s e i n pum p c on c e n t r a t i on i s de pe nde n t on r a p id p r o -t e in syn the s i s a nd se c r e t i on v i a t he G o lg i a ppa r a tu s(Hager e t a l . 1991) can be tes ted .F igu r e 1 A show s a t yp i c a l r e c o r d ing o f V M f r om a

A- 1 0 0>

E

- 1 2 0

15 m in

1 00 BM CHI

BM a t e r i a l a n d m e t h o d sC oleop t i l es wer e ob ta ined f r om 4- day- o ld , da r k - g r own cor n (Zeamays L . c v . M u t i n ; K W S S a a t z u c h t, E i n b e c k , F e d e r a l R e p u b l ic o fG e r m a n y ) s e e d l i n g s. S u b - a p i c a l c o l e o p t il e s e g m e n t s ( 1 0 m m l o n g )w e r e s e p a r a t e d f r o m t h e p r i m a r y l e a f a n d f r o m t h e c o | e o p t i l e a p e x( 5 r am) . Af t e r gen t ly ab r ad ing wi th d i a tomaceous ea r th co leop t i l esw e r e p r e q n c u b a t e d i n s t a n d a r d e x p e r i m e n t a l m e d i u m ( i n m M :0.1 KCI, 0.1 NaC1, 0.1 CaCla, 2 .5 Tr is /2 -(N-m orph ol ino ) etha nesu l-phon ic ac id ( M E S) ) f o r ~_1 h p r io r t o t he ex per ime n t i n o r der t odep le t e any endogenous aux in . For vo l t age r ecor d ings , co l eop t i l es e g m e n t s w e r e f i x e d h o r i z o n t a l l y in a n e x p e r i m e n t a l c h a m b e r a n dce l l s f r om the ou te r ep ider mis impaled wi th a conven t iona l micr o -e l e c tr o d e c o n t a i n i n g 3 0 0 m M K C t . T h e v o l t a g e d i ff e r e n c e b e t w e e nthe in t r ace l lu l a r ly pos i t i oned e l ec t r ode an d a r e f e r ence in t he ba thw a s m e a s u r e d w i t h a v o l t a g e a m p l i f e r (g i P ; W y e S c i e n ce , W y e ,U. K. ) . E longa t ion as s ays wei~e ca r r i ed ou t w i th ab r ad ed co leop t i l e

- 1 0 0

E - 1 1 o

- 1 2 0 t O0 gM CH IFig . 1 . A , B . I ndo l ace t i c ac id - evoked po la r i s a t ion and cyc lohex-i r n ide evoked d epo la r i s a t ion o f co l eop t i l e ce ll s . A An aux in - dep le t edand abr aded co leop t i l e was f i r s t ba thed in s t andar d buf f e r bef o r e10 uM I A A w as added . Af t e r r each ing a s t ab l e po la r i s ed VM in I AAt h e w o t e i n s y n t h e s is i n h i b it o r C H I ( 1 0 0 g M ) w a s a d d e d . B C H I ( 1 0 0b tM ) w a s a d d e d d i r e c tl y t o t h e s t a n d a rd b a t h i n g m e d i u m ( - I A A ) o fan aux in - dep le t ed co leop t i l e . Ar r ows ind ica t e add i t i on o f I AA andC I I I , r es pec t ive ly

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1 8 4 G . T h i e l e t a l .: M a i n t e n a n c e o f p o l a r is e d m e m b r a n e v o l t a g e

AVM2O

10 . 4 ;/

0l 10 0 I t I I40 60 80 100

CHI / gMF i g . 2 . M e a n i n h i b i to r - g e n e r a t e d m e m b r a n e d e p o l a r i sa t i o n ( A V M ) o fc o l e o p t i l e c e l l s a s a f u n c t i o n o f C H I c o n c e n t r a t i o n . F o u r a b r a d e dc o l e o p t i l e s w e r e k e p t i n s t a n d a r d e x p e r i m e n t a l m e d i u m c o n t a i n i n g1 0 g M t A A a n d c h a l l e n g e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f C H I .F i t t in g o f m e a n A V .~ d a t a _+ S E w i t h t h e M i c h a e l i s - M e n t e n e q u a t i o n( d o t t e d li n e ) y ie l d s a c o n c e n t r a t i o n o f 1 7 g M C H I f o r h a l f m a x i m a ldepo l a r i sa t i on

m a iz e c o l e op t i l e c e l l . I n s t a nda r d e xpe r im e n ta l m e d i -u m t h e r es t in g v o l t a g e w a s s t ab l e a t - 1 0 8 m V . A d d i n gI A A ( 1 0 ~ t M ) t o t h e b a th i n g m e d i u m c a u s e d t h e w e l lkno w n s low po la r i s a t i on o f V M ( F ig . 1 A ) . S ubse qu e n ta d m i n i s tr a ti o n o f C H I ( 1 0 0 a M ) e v o k e d a s l o w p o s i -t ive s h i f t of TV~, leve l l in g o f f , in the prese nt ex am ple ,a t a n e w s ta b le v o l ta g e o f - l l 0 m V . C o m p a r a b l er e su l t s w e r e ob ta ine d in a l l t he c e l l s t e s t e d a nd them e a n da t a a r e sum m a r i se d in Ta b le 1 . F igu r e 2 i l l u s -t r a te s t ha t t he e f f e c t o f C H I on V M w a s c onc e n t r a t i on -de pe nde n t , w i th a c onc e n t r a t i on o f 17 ~ tM C H Ir e qu i r e d f o r ha l f m a x im a l A VM .The m e a n ha l f t im e ( tl /2 ) o f C H I a c t ion , i . e. , t he t im eb e t w e e n a d d i t i o n o f 1 0 0 ~ t M C H I a n d h a l f m a x i m a lAVM, wa s 9 .8 + 0 .6 min (n = 8) . This i s ve ry s imi la rt o t h e C H I g e n e r a t e d d e c a y o f A T P a s e c o n c e n t ra t i o n( hn = 12 m in ; H a g e r e t a l. 1991 ) a nd som e w h a t f a s t e rt h a n C H I - i n d u c e d d e c a y o f t h e c o l e o p ti l e e l o n g a ti o nr a t e ( q/2 = 21 m in ; H a g e r e t a l . 1991 ) ob ta ine d inm a iz e c o l e op t i l e s unde r t he s a m e c ond i t i ons .T h e d e p o l a r i s i n g e f f e c t o f C H I w a s n o t d e p e n d e n t o np r e - t r e a tm e n t o f c o l e op t i l e s w i th I A A . F igu r e 1 Bs h o w s t h a t a d d i t i o n o f 1 0 0 ~ M C H I t o t h e b a th i n gm e d i u m o f a n a u x i n - d e p l et e d c o l e o p t i l e a l s o r e s u lt e din a s low ( tu2 = 8 .2 + 1 min , n = 4) p os i t iv e sh i f t ofVM f r o m - 1 1 3 t o - 1 0 3 . T h e a v e r a g e C H I - i n d u c e dAVM in auxin-dep le ted ce l l s i s sum mar ised in Table 1 .F o r t e n t a t i ve c ha r a c t e r i s a ti on o f V M in t he p r e se nc e o fC H I , c o l e op t i l e c e l l s w e r e i nc uba te d in s t a nda r de x p e r i m e n t a l m e d i u m c o n t ai n i n g 1 m M N a C N p l u s

- 8 0 ->I=: -120 -

_- 1 6 0 -

1 0 min

tO0 aMCCR 1 00 gMCH tF ig . 3 . In sen s i t i v i t y o f Vt4 o f t he IAA -t rea t ed co l eop t i l e ce l l t o RN A-s y n t h e s is i n h i b i t o r C O R . V M w a s s t a b l e i n t h e s t a n d a r d b a t h i n g m e d i -u m c o n t a i n i n g I A A a n d r e m a i n e d u n a f f e c t e d a f t e r a d d i n g 4 0 0 # MC O R . S u b s e q u e n t a d d i t io n o f C H I ( 1 0 0 g M ) e v o k e d d e p o l a r i s a t io no f VM. Arrow s i nd i ca t e add i t i on o f CO R an d CHI , re spec t i ve ly

> - 8 0 jE -12o

-1 60 f t20 mg/ I 100 ,uM

BF A CHIF ig . 4 . In se ns i t i v i t y o f VM o f an IA A-t rea t ed co l eop t i l e ce l l t o i nh ib i -t i on o f Go lg i -m ed ia t ed ves i c l e sec re t i on . VM o f t he co l eop t i l e ce l lw a s s t a b l e in t h e s t a n d a r d b a t h in g m e d i u m c o n t a i n i n g 1 0 v M I A Aa n d r e m a i n e d u n a f f e c t e d b y a d d i n g 2 0 m g / t B F A . S u b s e q u e n t a d d i -t i on o f CH I (100 l aM) evoked depo l a r i sa t i on o f VM. Arro ws i nd i ca t ea d d i t io n o f B F A a n d C H I , r e s p e c t i v e l y

0 . 4 m M sa l i c y lhyd r ox a m ic a c id t o i na c t iva t e H + -A TP a se by a r a p id m e ta bo l i c b loc k ( S l a ym a n e t a l.1973 , B la t t 1987 ) . I n c o l e op t i l e c e l l s t h i s p r oc e du r ec a u s e d r a p i d d e p o l a ri s a ti o n t o a b o u t - 1 0 0 m V ( d a tan o t s h o w n ) , c o m p a r a b l e t o t h e m e a n l / ~ , ~ r e c o r de d insa tu r a ti ng C H I ( Ta b le 1 ).C o r d y c e p i n , a n i n h i b it o r o f R N A s y n t h e s is ( D e l s e n ye t a l. 1975 ) , a l so r e ve r se s r a p id ly i n m a iz e c o l e op t i l e sa t 400 ~ tM I A A - s t im u la t e d e longa t ion ( tl /2 = 24 m in ;Ede lm a nn e t a l . 1989 : f i g 5 ) a nd a ux in e nha nc e dA TP a se c onc e n t r a t i on ( H a ge r e t a l . 1991 ) . The e f f e c to f C O R on VM w a s t e s t e d u s ing c o l e op t i l e c e l ls w i ths t a b l e po la r i s e d vo l t a ge s i n t he p r e se nc e o f 10 ~ MI A A . F i g u r e 3 s h o w s th a t a d d it i on o f 4 0 0 g M C O Rpr odu c e d no s ign i f i c a n t e f f e c t on V M ove r t he 45 m inr e c o r d ing p e r iod . The s a m e in se ns i t i v i t y o f V M toC O R t r e a tm e n t w a s obse r ve d in a l l f i ve c o l e op t i l e st e s t e d ( Ta b le 1 ). F igu r e 3 a l so show s tha t subse que n ta d d i t i o n o f C H I ( 1 0 0 F M ) i n t h e p r e s e n c e o f C O Rr e su l t e d in t he t yp i c a l s l o w pos i t i ve sh i f t o f V M p r o -duc e d b y the f o r m e r i nh ib i to r ( c f . F ig . 1 ). Th i s show s

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G . T h i e l e t a l.: M a i n t e n a n c e o f p o l a r i s e d m e m b r a n e v o l t a g eT a b l e 1 . E f fe c t o f I A A a n d i n h i b i to r s o n m e m b r a n e v o l t a g e o f m a i z e c o l e o p t i le c e l l s

I 8 5

M e a n V u M e a n AV Mdc o n t ro P C H I u N a C N + S H A M ~ B F A C O R- I A A + I A A - I A A + I A A - I A A 3 0 r a in 4 5 r a in 3 0 m i n 4 5 r a in

- 1 1 6 - 1 3 5 _+ 2 ( 2 2) - 9 9 _ + 4 ( 4 ) - 1 0 7 _ + 6( 8 ) - 9 7 0 . 2 5 + 2 ( 8 ) 1 . 2 5 + 2 ( 6 ) - 3 + 2 ( 5 ) - 3 _ + I ( 3 )10 0.6 22 3 .3

M e an con t r o l VM in s t andar d exper im en ta l s o lu t ion bef o r e ( - I AA ) and a f t e r max im al po ta r i s a t ion in 10 ~ tM I AA ( +I AA )b M e an s t ab le VM and ( AVM be low) a ch ieved a f t e r add i t i on o f 100 gM C H I to s t andar d s o lu t ion wi th o r wi thou t 10 , aM I AA

S t a b l e VM a f t e r a d d i n g 1 m M N a C N a n d 0 . 4 m M s a l i c y lh y d r o x a m i c a c i d ( S H A M ) t o s t a n d ar d e x p e r i m e n t a l s o lu t i o n ( - I A A )M e a n A VM o b t a i n e d 3 0 r a i n a n d 4 5 r a i n a f t e r a d d i n g 2 0 r ag /1 B F A o r 4 0 0 g M C O R t o s t a n d a rd e x p e r i m e n t a l s o lu t i o n c o n ta i n i n g 1 0 u M I A A

VM va lues g iven in m V of n exper ime n t s ( i n b r acke t s ) as m ean _+ SE

0. 1

i ! i0 45 90

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EE

0

0 3o

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Fig 5 . E longa t ion o f ab r aded co leop t i l e s egment s i n t he p r es ence o f20 g M I A A wi thou t ( 9 con t r o l ) , wi th add i t i on o f B F A ( Q, 20 r ag / l ) ,C O R ( ~ 5 , 4 0 0 ~ t M ) a n d C H I ( 1 1 , 1 0 0 g M ) . A u x i n a n d i n h i b it o r s w e r eadded a t t ime zer o . Da ta po in t s a r e means o f 3 ba t ches o f 5 co leop-t i l e s egment s

t ha t a bse nc e o f a V M r e sponse to C O R i s no t due tol im i t e d a c c e s s ib i l i t y o f t he im pa le d c e l l t o e x t r a c e l lu -la r ly appl ied inhib i tor s .I f IA A - s t im u la t e d a c t iv i t y o f t he H +- A TP a se r e qu i r e dsyn the s i s o f sho r t - l i ve d A T P a se p r o t e in s ( H a ge r e t a l.1991 ) o r r e gu la to r y p r o t e in s ( C ho a nd H ong 1995) ,t he c on t r ibu t ion o f t he H +- pum p to V M m u s t bede c r e a se d by inh ib i to r s o f i n t r a ce l lu l a r ve s i c l e t r a f fi c .To t e s t t h i s pos tu l a t e d r e l a t i onsh ip , c o l e op t i l e s w e r et r e a t e d w i th B F A , a n e f f e c t ive i nh ib i to r o f G o lg i -m e d ia t e d ve s i c l e t r a nspo r t i n c o l e op t i l e s ( S c h ind le r e ta l . 1994 , Ede lm a nn a nd V o lkm a nn 1996) . F igu r e 4s h o w s a n e x a m p l e i n w h i c h 2 0 ra g /1 B F A w a s a d d e dto t he ba th ing m e d ium o f a n a b r a de d c o l e op t i l e . A tth i s c onc e n t r a t i on B F A r a p id ly a nd se ve r e ly i nh ib i t sc o l e op t i l e e longa t ion ( S c h ind le r e t a t. t 994 : f i g . 5 )a nd the g r a v i t r op ic r e sponse ( Ede lm a nn a nd V o lk -

m a nn 1996) , bu t t he i nh ib i to r ha d no s ign i f i c a n tim pa c t on ~ t~ ove r t he 45 r a in r e c o r d ing pe r iod . Thesa m e in se ns i t i v i ty o f VM tO 20 r a g/1 B F A w a s f ound inse ve n c om pa r a b le e xpe r im e n t s ( Ta b le 1 ) . F igu r e 4a l so show s tha t a dd ing 100 #M C H I a s w e l l a s B F Ar e su l t e d in the c ha r a c t er i s t ic C H I - induc e d de po la r i s a -t i on . Th i s a ga in show s tha t e x t r a c e l lu l a r ly a pp l i e dinh ib i to r s a r e a c c e s s ib l e t o t he im pa le d c e l l .To r e l a t e t he e f f e c t o f i nd iv idua l i nh ib i to r s on V M tothe i r im pa c t on c o l e op t i l e e longa t ion , t he l a t t e r w a sm e a su r e d in t he p r e se nc e o f the va r ious i nh ib i to r s.F igu r e 5 show s a ux in - e nha nc e d c o l e op t i l e e longa t ionin t he p r e se nc e a nd a bse nc e o f i nh ib i to r s . To a l l owc om p a r i son be tw e e n V M a nd e longa t ion m e a s u r e -m e r it s , c o l e op t i l e s e gm e n t s w e r e a l so a b r a de d a nd theinh ib i to r s a dde d a t t he s a m e c onc e n t r a t i on a s f o r V Mr e c o r d ings . F igu r e 5 show s tha t a ll t h r e e d r ugs i nh ib i tc o l e op t i l e e longa t ion ; C O R a nd C H I c a use d r a s t i cinh ib i t i on , B F A m ode r a t e i nh ib i t i on . The e f f e c t o find iv idua l i nh ib i to r s f o r 45 r a in pe r iods on V M d id no tc o r r e l a t e w i th t he i r im pa c t on c o l e op t i l e e longa t ion(F ig . 4 and Table 1).Di s cu s s i on

I t i s ge ne r a l ly a c c e p te d tha t t he a ux in - ge ne r a t e dh y p e r p o l a ri s a ti o n o f t he m e m b r a n e i n m a i z e c o l e o p -t i ie ce l l s i s due to an inc rease in H+-ATPase ac t iv i ty( r e v i e w e d in N a p ie r a nd V e n i s 1995 ) . A ga ins t t h i sba c kg r ound , l /~ i r e c o r d ings i n i n t a c t c o l e op t i t e c e l l sc a n be s e e n a s a s im p le i nd i r e c t a s sa y o f c ha nge s i npum p a c t iv i t y . The a im o f the p r e se n t i nve s t iga t ionw a s to t e s t i n v ivo w he the r t he a ux in - s t im u la t e d r i s ein A TP a se a c t iv i t y i s due to ho r m one - e nha nc e d syn -the s i s a nd inc o r po r a t ion o f A TP a se m o le c u le s i n to t hep la sm a m e m br a ne ( H a ge r e t al . 1991 ) o r t o ho r m one -e nha nc e d syn the s i s a nd se c r e t i on o f a sho r t - l i ve d r e g -

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186 G , Th i e l e t a l .: Ma in t enanc e o f po l a r i sed m em bran e vo l t ageu la to r y p r o t e in ( La do e t a l. 1977 , C ho a nd H ong1995) w h ic h m igh t c on t r o l A TP a se a c t iv i t y .The p r e se n t vo l t a ge r e c o r d ings c on f l i c t w i th t h i shypo the s i s i n s e ve r a l ke y a spe c t s : f i r s t , t he p r o t e insyn the s i s i nh ib i to r C H I r e ve r se s t he I A A - induc e d r i s ein A TP a se c onc e n t r a t i on ( H a ge r e t a l . 1991 ) a ndshou ld the r e f o r e on ly r e ve r se t he I A A - ge ne r a t e dhype r po la r i s a t i on . H ow e ve r , t he i nh ib i to r c a use dm e m br a ne d e po la r i s a t i ons i r r e spe c t ive o f t he p r e s -e nc e o f a ux in ( s e e a l so La do e t a l. 1977 ) . Thus , C H Im u s t a f f e c t c o n s t i t u t i v e p r o p e r t i e s o f m e m b r a n et r a nspo r t un r e l a t e d to a ny I A A - ge ne r a t e d r i s e i nA TP a se c onc e n t r a t i on ( H a g e r e t a l. 1991 ) o r o the rI A A - d e p e n d e n t r e g u l a t o r y p r o t e i n s ( C h o a n d H o n g1995) . S e c ond ly , t he m e m br a ne vo l t a ge in a ux in -t r e a t e d c o l e op t i l e c e l l s i s no t a f f e c t e d by B F A , t he l a t-t e r be ing a po te n t i nh ib i to r o f ve s i c l e t r a nspo r t i ng r a s s c o l e op t i l e s ( S c h ind le r e t a l. 1994 , Ede lm a nna nd V o tkm a nn 1996) unde r t he se c ond i t i ons . The r e -f o r e , i t i s un l ike ly t ha t t he a ux in - s t im u la t e d hype r po -l a r i s a t i on i s due to ho r m one - c on t r o l l e d syn the s i s o fA TP a se m o le c u le s ( H a ge r e t a l. 1991 ) o r p r o t e in sw h ic h r e gu la t e A TP a se a c t iv i t y ( C ho a nd H ong .1995 ).T h e i r c o n t r ib u t i o n t o t h e m e m b r a n e v o l t a g e s h o u l d b eina c t iva t e d w he n G o lg i - m e d ia t e d ve s i c l e t r a f f i c , t hege ne r a l pa thw a y f o r p r o t e in s t r a nspo r t f r om the ER tothe p l a sm a m e m br a ne in e uka r yo t i c c e l l s ( S t a e he l ina nd M oor e 1995 ) , i s b loc ke d by B F A . Th i r d ly , t heR N A s y n t h e s i s i n h i b i t o r C O R r e v e r s e s t h e I A A -induc e d r i s e i n A T P a se a c t iv i t y ( H a ge r e t a l . 1991 ) , a ne f f e c t a t t r i bu t e d to t he c on t inuous syn the s i s o fA T P a se m o le c u le s ( H a ge r e t a l. 1991 ) o r p r o t e in sr e qu i r e d f o r H +- A TP a se r e gu la t i on ( C ho a nd H ong1995) . S inc e e qu iva l e n t t r e a tm e n t o f c o l e op t i l e s w i thC O R p r odu c e d n o sho r t - t e rm e f f e c t on l /~ u, c o r r e l a -t io n b e t w e e n c o n s t i tu t i v e o r I A A - p r o m o t e d t ra n s c ri p -t i o n w i t h m e m b r a n e t r a n s p o r t p r o c e s s e s c a n b e d i s -c oun te d .I n sum m a r y , t he vo l t a ge r e c o r d ings show tha t a ux in -de pe nde n t A TP a se - a nd ge ne r a l p r o t e in syn the s i sa nd /o r de l ive r y t h r ough the s e c r e to r y pa th w a y i s no ts ign i f i c a n t f o r the m a in t e na nc e o f a n a ux in - de pe nde n thype r po la r i s a t i on in t he sho r t t e r m . Thus , t he ho r -m one - s t im u la t e d r i s e i n pum p a c t iv i t y , t he p r oc e s sunde r ly ing hype r po la r i s a t i on , m us t be due to k ine t i cm o d u l a t i o n o f t h e A T P a s e . T h e l a tt e r m u s t o c c u r v i a am e c h a n i sm tha t doe s no t i nvo lve a sho r t - l ive d r e gu la -to r y p r o t e in .I n t e r p r e t a ti on o f the p r e se n t da t a i s in a g r e e m e n t w i ththe f i nd ing tha t I A A m od i f i e d the t r a nsc r ip t ion l e ve lo f P - t y p e A T P a s e s i n t o m a t o h y p o c o t y l , b u t t h e

m o du la t e d t r a nsc rip t ion l e ve l w a s no t c o r r e l a t e d w i tht h e ti m e c o u r s e o f I A A - i n d u c e d g r o w t h ( E w i n g a n dB e nne t t 1994 ) . The p r e se n t da t a a r e a l so i n a c c o r -da nc e w i th r e c e n t i nve s t iga t ions r e po r t i ng a l a c k o fa ux in - s t im u la t e d r i s e i n A TP a se c onc e n t r a t i on inp l a sm a m e m br a ne f r a c t ions f r om m a iz e c o l e op t i l e s( J a hn e t a l. 1996 ) o r sun f lo w e r hypo c o ty l s ( C h o a ndH ong 1995) . J a hn a nd c o - w or ke r s ( 1996 ) spe c u la t e dtha t t he l a c k o f a de t e c t a b l e r i s e in A T P a se c onc e n t r a -t i on in f r a c t iona t e d m e m br a ne p r e pa r a t ions m igh t bedue to the fac t tha t the la t te r i s conf ined to oute r ep i -de r m a l c e l ls . I n th i s c a se t he e f f e c t m a y be m a ske d b ythe bu lk f ra c t ion o f c o r t i c a l c e l l m e m br a ne s . H ow e v-e r , i n t he p r e se n t s tudy da t a w e r e ob ta ine d f r om theou te r e p ide r m is o f i n ta c t c o l e op t i t e c e l l s w h ic h m a ke si t un l ike ly , t ha t a n a ux in - ge ne r a t e d r i s e i n A TP a sec onc e n t r a t i on i s c on f ine d to t he e p ide r m a l t i s sue .O ne in t e r es t i ng r e su l t o f t he p r e se n t s tudy w a s t ha tC H I e v o k e d d e c a y i n I A A - e n h a n c e d e l o n g a ti o n ( e . g. ,H a ge r e t al . t 99 1 ) a nd de c r e a se i n V M ( F ig. t ) w i th as t r i k ing ly s im i l a r k ine t i c s . The c lo se t e m por a l c o r r e -l a t ion o f bo th pa r a m e te r s sugge s t s t ha t t he C H I -e voke d de po la r i s a t i on i s r e l a t e d to t he C H I - e voke dinh ib i ti on o f c o l e op t i l e e longa t ion . A p l a us ib l e e xp la -na t ion f o r suc h a l i nk i s t ha t t he a n t ib io t i c - e voke dde po la r i s a t i on a bo l i she s K + up ta ke th r ough inw a r dr e c t i f y ing K + cha nne l s , a p r oc e s s be l i e ve d to bee s se n t i a l f o r t u r go r d r ive n g r ow th ( Th ie l e t a l. 1996 ) .I n a dd i t i on , a m or e po s i t i ve V M c ou ld r e v e r se t he d r iv -ing f o r c e f o r pa s s iv e K + f lux , r e su l t ing in t he l e a ka g eo f o s m o l y t e s f r o m C H I - t r e a t e d m a i z e c o t e o p t i l e s( K u t sc he r a a nd S c hopf e r 1986 ) . I n t h i s c on te x t t heinh ib i to r y e f f e c t o f C H I on p r o t e in syn the s i s ( Ede t -m a n n e t al . t 9 8 9 ) m a y b e v i e w e d a s co m p l e m e n t a r yto t he i nh ib i t ion o f c o l e op t i l e e longa t ion a t l e a s t in t hesho r t t e r m .T h e m e c h a n i s m b y w h i c h C H I c a u s e s m e m b r a n ede po la r i s a t i on r e m a ins unknow n . The s im i l a r i t ybe tw e e n I~M va lue s o b ta ine d f r om C H I - t r e a t e d a ndm e ta b o l i c a l ly b loc k e d c e i l s sugge s t s t ha t C H I inh ib i t so r sho r t - c i r c u i ts t he c on t r ibu t ion o f H + - A TP a se to ~ v~ .G iv e n tha t B F A h a d no sho r t - t er m e f f e c t on V M, i t c a nb e c o n c l u d e d t h a t C H I - e v o k e d d e p o l a r i s a t i o n i s n o tc a u s e d b y t h e i n h ib i ti o n o f n e w l y s y n t h e s i s ed p r o -t e in s. The l a t te r w o u ld no t r e a c h the i r s i te o f a c t i on ont h e p l as m a m e m b r a n e i n t he p r e s e n c e o f B F A . T h e r e -f o r e , t he da t a sugge s t t ha t t he a n t ib io t i c doe s no t a c tspe c i f i c a l l y a s a n i nh ib ito r o f p r o t e in syn the s i s b u tp r oduc e s s ign i f i c a n t s ide e f f e c t s w h ic h ha ve o f t e nbe e n r e po r t e d in t he pa s t ( E l l is a nd M a c D ona ld 1970 ,M c M a h o n 1 9 7 5 ) .

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G. T h ie l e t a l .: M ain tenan ce o f po lax i s ed mem br ane vo l t age 18 7A c c o r d i n g t o t h e " a c i d g r o w t h t h e o r y " , c e l l e l o n g a -t i o n i s a c h i e v e d b y a u x i n - s ti m u l a t e d p r o t o n e x t r u s io na n d c o n s e q u e n t l o o s e n i n g o f c e l l w a l l s t r u c t u r e s ( e . g . ,R a y l e a n d C l e l a n d 1 9 9 2 ) . I t is i n t e r e st i n g t o n o t e t h a tC O R a n d B F A , i n h i b i to r s w h i c h r e d u c e I A A -e n h a n c e d H + e x t r u s i o n ( H a g e r e t a l. 1 9 9 1 , S c h i n d l e re t a l. 1 9 9 4 , C h o a n d H o n g 1 9 9 5 ) , h a v e n o s ig n i f i c a n ti m p a c t o n VM . T h u s i t m u s t b e c o n c l u d e d t h at I A A -s t i m u l a t e d H + s e c r e t i o n i s n o t a c h i e v e d s i m p l y b ya u x i n - d e p e n d e n t s t im u l a t i o n o f t h e H + - A T P a s e. T h e r ei s c l e a r l y a n o b l i g a t o r y r e q u i r e m e n t f o r a f u n c t i o n a ls e c re t o ry s y s t e m t o g u a ra n t ee c o m p l e t e I A A - p r o m o t -e d H + e x t r u s i o n ( S c h i n d l e r e t a l . 1 9 9 4 , C h o a n d H o n g1 9 9 5 ) . T h i s c o r r e la t i o n i s r e m i n i s c e n t o f a h y p o t h e s i ss u g g e s t e d b y R a y ( 1 9 7 7 ) , n a m e l y t ha t I A A - e v o k e da c i d i f i c a t i o n o f t h e c e l l w a l l i s n o t A T P a s e - m e d i a t e db u t r e s u l t s f r o m s e c r e t i o n o f E R - d e r i v e d a c i d i cv e s i c l e s i n t o t h e a p o p l a s t . W h e t h e r t h i s p u t a t i v e p r o -t o n s e cr e t io n i s i n d e e d t h e m e c h a n i s m w h i c h i n d u c e sg r o w t h o r o n l y a n e p i p h e n o m e n o n n e e d s f u r t h e ri n v e s t i g a t i o n .A c k n o w l e d g e m e n t sT h e w o r k w a s s u p p o r t e d b y t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t( SFB 523) . W e a r e g r a t e f u l to Dr . J u l i a Dav ies ( Unive r s i ty o f Yor k)f o r h e t p w i t h t h e m a n u s c r i p t.

R e f e r e n c e sB ates G, C le l and R E ( 1979) P r o te in s yn thes i s and aux in - inducedg r o w t h : i n h i b i to r s tu d i e s. P l a n t a I 4 5 : 4 3 7 - 4 4 2B la t t M R ( 1987) E lec t r i ca l char ac t e r i s t i cs o f s tomata l guar d ce l l s :

t h e c o n t r i b u t i o n o f A T P - d e p e n d e n t , " e l e c t r o g e n i c " t r a n s p o r tr evea led by cur r en t - vo l t age and d i f f e r ence- cur r en t vo l t agea n a l y s is . J M e m b r B i o l 9 8 : 2 5 7 - 2 7 4

C h o H - T , H o n g Y - N ( 1 9 9 5 ) E f fe c t o f I A A o n s y n t h e s i s a n d a c t i v i tyo f t h e p l a s m a m e m b r a n e H + - A T P a s e o f s u n f l o w e r h y p o c o t y ls , i nr e l a t ion to I AA - induc ed ce l l e longa t ion and H + excr e t ion . J P l an tP h y s i o l i 4 5 : 7 1 7 - 7 2 5

C u n n i n g h a m e M E , H a l l J L ( 1 9 8 6 ) T h e e f f e c t o f c a lc i u m a n t a g o n i s t sand inh ib i to r s o f s ec r e to r y p r oces s es on aux in - induced e longa-t ion and f ine s t r uc tu r e o f Pisum sativunz s t em s egment s . P r o to -p l a sm a 1 3 3 : 1 4 9 - 1 5 9

D e l s e n y M , P e r a l ta M T , G u i t t o n Y ( 1 9 7 5 ) E f f e c t o f c o r d y ce p i n o nR N A m e t a b o l i s m i n g e r m i n a t i n g s e e d l i n g s . B i o c h e m B i o p h y sR e s C o m m u n 6 4 : 1 2 7 8 - 1 2 8 5

E d e t m a n n H I S c h o p f e r P ( 1 9 8 9 ) R o l e o f p r o t e i n a n d R N A s y n t h e s isin the in i t i a t i on o f aux in - media t ed g r owth in co teop t i l es o f Zeamays L . P l a n t a 1 7 9 : 4 7 5 - 4 8 5

- V o l k m a n n D (1 9 9 6 ) T h e e f f e c t o f b r e f e l d i n A o n th e re d i s tr i b u -t ion o f os mophi l i c par t i c l es and the g r av i t r op ic r es pons e o f r yeco leop t i l es . P r o top las r na 190 :1 - 7

E l l i s R J , M a cDo nald I R ( 19 70) Spec i f i c i ty o f cyc lohex im ide in h igh-e r p l a n ts . P l a n t P h y s io l 4 6 : 2 2 7 - 2 3 2

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Fr i as I , C a lde i r a M T , P er ez- C as t ine i r a J R , Navar r o - A vino J P , C u l i a -n e z - M a c i a F A , K u p p i n g e r O , S t r a n s k y H , M o n t s e r r a t P , H a g e rA , S e r r a n o R ( 1 9 9 6 ) A m a j o r i s o f o n n o f th e m a i z e p l a s m a m e m -b r a n e A T P a s e : c h a r a c t e ri z a t io n a n d i n d u c t i o n b y a u x i n i n c o I e -o p t il e s . P l a n t C e l l 8 : i 5 3 3 - 1 5 4 4

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