391

Biochimica et Biophysica Acta, 4 0 2 ( 1 9 7 5 ) 3 9 1 - - 4 0 2 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98397

H O R M O N A L C O N T R O L OF T R A N S C R I P T I O N IN H I G H E R PLANTS

M A R C E L TEISSERE, P A U L PENON, R O B E R T B. V A N HUYSTEE*, Y A N N I C K A Z O U and J A C Q U E S R I C A R D Laboratoire de Physiologie Cellulaire Vdgdtale Associd au C.N.R.S., Universitd d'Aix-Marseille II, U E R de Luminy, 70, route Ldon Lachamp, 13288 Marseille Cedex 2 (France)

(Received February 3rd, 1975)

Summary 1. Nucleolar R N A polymerase Ib obtained from auxin-treated lentil roots exhibits a higher transcriptional activity than the enzyme obtained from control roots. This difference is due to a change in the enzyme properties after auxin treatment. It is suggested that the hormonal effect is mediated b y a factor that changes the molecular properties of nucleolar R N A polymerase. 2. Four fractions, ~, fl, 7 and 5, that stimulate the activity of R N A polymerase Ib, have been extracted from lentil roots. Two of them, 7 and 8 have been studied. Factor 5 can stimulate nucleolar polymerase Ib and the nucleoplasmic e n z y m e II equally well, while factor 7 is specific for polymerase Ib. 3. The curve of UMP incorporation in vitro, with and without factors 7 or suggests that they are initiation factors. This conclusion is reinforced by the analysis of simultaneous incorporation of [7- 3 2 p] ATP and [ 3 H] UMP in the RNAs synthesized in vitro. 4. Although the level of factor 5 is independent of auxin treatment, that of factor 7 is doubled in auxin-treated roots. These results suggest that factor "y is an auxin-induced protein that modulates the specific activity of the nucleolar R N A polymerase. 5. A general model of the mode of action of auxins at the molecular level is proposed. It integrates into a unified scheme the above results as well as those obtained b y other workers.

* On sabbatical leave from the d e p a r t m e n t of Plant Sciences, University of Western Ontario, London, Ontario, Canada.

392 Introduction The elucidation of the nature of hormonal control of plant growth, at the molecular level, is still an appealing challenge. In recent years, it was conclusively shown t h a t auxins control independently both cell wall extension [1--3] and the transcription of DNA [4,5]. We have shown [6,7] that when a plant tissue is fed with the hormone indole 3-acetate, two types of events occur depending on the length of the hormonal treatment. With 'short' (1 h or less) treatments, the auxin increases the transcriptional availability of the isolated chromatin [6], leading to the synthesis of both heterogeneous nuclear RNAs and short-lived messenger RNAs [7]. Although short treatments do not modify the level of nucleolar and nucleoplasmic transcriptases, longer treatments {more than 14 h) double the level of nucleolar polymerases leaving that of nucleoplasmic enzymes unchanged [6]. Moreover, it was established that the increased level of the nucleolar enzymes is n o t due to their synthesis, but to a dramatic change in their molecular properties [6]. This conclusion was confirmed later [8] with a different auxin: 2,4-dichlorophenoxy acetate. These results clearly suggest that the change could be the consequence of the association of the enzyme with a protein factor, the synthesis of which could be under the control of the hormone. The present paper gives direct experimental evidence for the existence of this transcription factor, as well as information as to its mode of action. Material and Methods The lentil seeds (Lens culinaris vat. plate blonde) were germinated as previously described [6]. After three days the roots reached a length of about 3 cm and were harvested. Auxin treatment (indole 3-acetate, 2.5 • 10 -4 M) was effected 20 h prior to harvesting. Nuclei were isolated following a technique derived from that of Mertelsmann [9]. About 100 g of lentil roots were chilled for 15 min in ice water, and homogenized in a medium containing 100 mM Tris • HC1 pH 7.8, 350 mM sucrose, 15 mM magnesium acetate, 1 mM MnC12, 0.25 mM spermine and 12.5 mM 2-mercaptoethanol. The roots were homogenized in a Waring blendor, filtered through Miracloth at about 2°C, and centrifuged for 30 min at 2000 X g. The supernatant was discarded, and the pellet resuspended with a Potter-Thomas homogenizer in 25 ml of the grinding medium containing 0.5% w/v Triton X-100. After centrifugation the pellet contains the nuclear material. The nuclear protein fractions were obtained from this pellet following a technique derived from that of Alberga et al. [10]. The nuclear material, suspended in a medium containing 50 mM Tris " HC1 pH 7.4, 5 mM MgCI:, 5 mM dithiothreitol, was submitted to gentle magnetic stirring for 30 min at 4°C. After centrifugation for 10 min at 2000 X g, the supernatant was saved and the pellet submitted again to stirring in the above medium. This procedure was repeated three times. The collected supernatants contain the soluble nuclear proteins, and are defined here as the 'nuclear washings'. After the removal of soluble nuclear proteins, the pellet which consists of

393 deoxyribonucleoproteins and 'residual' proteins is defined as chromatin. The chromatin thus obtained was dispersed in 6 ml 10 mM Tris " HC1 pH 7.4, 10 mM (NH4)2SO4, 6 mM MgC12, 1 mM EDTA, 0.25 mM spermine, 5 mM dithiothreitol, 30% glycerol (v/v), and homogenized with a Potter-Thomas homogenizer. NaC1 was then added to obtain a final concentration of 1 M. The extract was then submitted to gentle magnetic stirring for 2 h at 4 ° C, and centrifuged for 30 min at 47 000 × g. The supernatant, which consisted of the free DNA, histories and non-histone proteins, was kept and dialyzed overnight against 250 ml of 10 mM Tris • HC1 pH 7.8, 1.5 mM EDTA, 2.5 mM dithiothreitol, 25% glycerol (v/v). The nucleohistones formed were eliminated by centrifugation (30 min at 47 000 X g). The supernatant contained the bulk of the non-histone chromosomal proteins. RNA polymerase Ib and the transcription factors were isolated from this supernatant. High recovery of RNA polymerase is obtained with this m e t h o d avoiding the sonication step in the presence of (NH4}~SO4 [ 1 1 ] . A small fraction {about 10%) of the initial activity remains associated with the nucleohistone pellet in either the 'control' or 'auxin-treated' preparation. The non-histone chromosomal proteins were loaded onto a carboxymethyl Sephadex C-25 (0.7 cm X 12 cm) column previously equilibrated with standard buffer (50 mM Tris " HC1 pH 7.8, glycerol 25% v/v, 5 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol). The column was washed with 5 ml standard buffer. The run off and the washing buffer were saved for isolation of R N A polymerase Ib. This was done following a technique already described [6]. The proteins b o u n d to the column are eluted stepwise as described in the legend of Fig. 2. Transcription stimulatory activity of the various protein fractions was determined b y measuring RNA polymerase Ib activity in the presence of these fractions. The polymerase activity was assayed by following the conversion of [3H] UTP to acid-insoluble material. The standard reaction mixture (0.2 ml) contained 39 mM Tris " HCI pH 7.8, 1.1 mM MnCI~, 1.5 mM KC1, 1.1 mM dithiothreitol, 1 mM phosphoenolpyruvate, 0.5 mM each of ATP, CTP, GTP, 0.0385 mM unlabeled UTP, 4 pCi [ 3H] UTP (20 Ci/mmol, Radiochemical Centre, Amersham), 2 pg pyruvate kinase (Sigma), 5 pg calf thymus DNA (Calbiochem) and the R N A polymerase. The reaction was run for 30 min at 35°C, and was stopped by adding 50 pl of a solution containing 80 mM sodium pyrophosphate and 40 pg of yeast RNA, then 2 ml of ice-cold 10% trichloroacetic acid. The reaction mixture was allowed to stand for 15 min in an ice-bath; the [3 H] UTP not incorporated was removed by centrifugation and repeated washings of the insoluble precipitate with 40 mM sodium pyrophosphate plus 5% trichloroacetic acid. The acid-insoluble RNAs were hydrolyzed for 20 min at 95°C, and the whole hydrolysate was p u t into vials containing 8 ml of Instagel (Packard) and counted in a Beckman LS 150 liquid scintillator. To test the deoxyribonuclease activity of the isolated protein fractions, calf t h y m u s DNA was incubated with either the protein fractions or a buffer for 30 min at 35 °C. The mixtures were then deproteinized by two chloroform extractions and filtered through a Sephadex G-75 column (0.7 cm × 20 cm) equilibrated with standard buffer. The eluted DNA was assayed for its template activity in a standard incubation mixture. The R N A chain-initiation studies were conducted as described by Seifart

394

et al. [ 1 2 ] , by measuring the simultaneous incorporation of 0.0066 mM (15 Ci/mmole, Radiochemical Centre, Amersham) of either [7-32P]ATP or [7.32p] GTP in the presence of [3H] UTP. In these cases the total concentration of either ATP or GTP was reduced to 0.05 mM in the incubation mixture, thus bringing the specific activity up to 1500 dpm per pmol. Phosphoenolpyruvate and pyruvate kinase were omitted in order to reduce non-specific transfer of phosphate. The RNAs synthesized were extracted from the reaction mixture first by buffer-saturated phenol, then by chloroform. The RNAs thus extracted were filtered through a Sephadex G-75 column (0.7 × 20 cm) equilibrated with a buffer containing 50 mM Tris • HC1 pH 7.8, 0.25 mM EDTA, 5 mM 2-mercaptoethanol, 20% glycerol (v/v). Filtration was performed at a flow rate of 6 ml/h. Fractions of 0.25 ml were collected, hydrolyzed for 30 min at 95°C in 5% trichloroacetic acid and counted in a Beckman LS 150 counter. Results

1. Existence of factors stimulating RNA polymerase Ib Salt sensitivity of purified nucleolar R N A polymerase Ib depends markedly u p o n whether or n o t the enzyme was extracted from auxin-treated tissues. Sensitivity to (NH4)2SO4 is greater with the enzyme obtained from treated roots. High salt concentrations normalize its specific activity to that of control enzyme. These results are exemplified in Fig. 1. The simplest interpretation of these data is that high ionic strengths result in the dissociation of a 'factor' from the 'treated' enzyme which then becomes identical to the 'control' e:izyme. This interpretation is n o t inconsistent with the fact that R N A polymerase preparations have been exposed to 1 M NaC1 during extraction. One needs to assume that the binding of the factor to the enzyme is reversible.

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395 TABLE I EFFECTS OF ~NUCLEAR W A S H I N G S ' A N D ' R U N - O F F ' F R A C T I O N S , FROM A U X I N - T R E A T E D T I S S U E S , ON T H E A C T I V I T Y O F R N A P O L Y M E R A S E I b

CONTROL

AND

T h e a c t i v i t y o f a f i x e d a m o u n t o f n u c l e o l a r R N A p o l y m e r a s e I b o b t a i n e d f r o m lentil r o o t s w a s a s s a y e d in t h e p r e s e n c e of 4 0 pl o f e a c h f r a c t i o n . Fraction added to e n z y m e Ib

UMP i n c o r p o r a t e d ( p m o l )

S t i m u l a t i o n (%)

None Nuclear Nuclear Run off Run off

2.94 3.85 4.02 3.76 4.45

31 37 28 52

w a s h i n g s f r o m c o n t r o l tissues w a s h i n g s f r o m a u x i n - t r e a t e d tissues f r o m c o n t r o l tissues f r o m a u x i n - t r e a t e d tissues

± 0.17 ± 0.23 +- 0 . 3 8 ± 0.27 ± 0.29

The idea that some 'factor' can bind to the nucleolar polymerase, or conversely dissociate from its surface, depending on the experimental conditions, gains further support from the results of Table I. Nuclear washings, which by themselves have no polymerase activity, significantly stimulate the activity of R N A polymerase Ib. When chromosomal acidic proteins are chromatographed on a DEAE-Sephadex column, the R N A polymerases are b o u n d to the column at low ionic strength, while the run-off fractions stimulate the polymerase Ib. The stimulatory activity of the run-off is more pronounced if the chromosomal proteins are extracted from auxin-treated and unwashed nuclei {Table I). The stimulatory fractions can be isolated. The non-histone chromosomal proteins obtained either from auxin-treated or from control tissues are passed over c a r b o x y m e t h y l Sephadex C-25 columns. The R N A polymerase activity appears in the run-off fraction while the stimulatory factors remain on the column. They can be eluted stepwise with KC1 in the standard buffer (Fig. 2). In this way, four stimulatory fractions, a, fi, 7, 5, are obtained. Fraction ~ is eluted b y the standard buffer with 43 mM KC1 and stimulates the activity of polymerase by a b o u t 90%. Fractions fi and 7 are eluted by 70 mM KC1 in the standard buffer, and they stimulate the polymerase activity b y a b o u t 90%. Fraction 5 is eluted b y 210 mM KCI in the same buffer and its stimulatory activity is considerable (700%). A highly inhibitory fraction is eluted immediately after 5. No a t t e m p t has been made to characterize this fraction. Whereas the level of fractions a, ~ and 5 are nearly the same in control and auxin-treated tissues, that of fraction 7, after auxin-treatment, is a b o u t twice {200%) that of the control.

2. Properties o f fractions 7 and 5 We have tried to gain further information on the nature and properties of active fractions 7 and 5 in an a t t e m p t to determine whether they are true transcription factors. Active fractions 7 and 5 are certainly proteins, since their stimulatory activity falls dramatically after preincubating with Enzite protease {Table IIa). The stimulatory activity of fraction 7 is almost completely suppressed by protease treatment; that of fraction 5 is not, unless the fraction is slightly heated

396

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Fig. 2. C & r b o x y m e t h y l - S e p h a d e x c h r o m a t o g r a p h y o f n o n - h i s t o n e p r o t e i n s f r o m c o n t r o l and a u x i n - t r e a t e d lentil r o o t s . A s a m p l e o f 5 . 5 m l c o n t a i n i n g 7.3 m g n o n - h i s t o n e p r o t e i n s e x t r a c t e d as d e s c r i b e d w a s l o a d e d o n t o a ( 0 . 7 c m × 1 0 c m ) c a x b o x y m e t h y l - S e p h a d e x C - 2 5 c o l u m n e q u i l i b r a t e d w i t h standard b u f f e r . T h e c o l u m n was w a s h e d w i t h 5 m l standard b u f f e r , t h e n t h e p r o t e i n s w e r e e l u t e d w i t h b u f f e r c o n t a i n i n g 43 raM, 7 0 m M a n d 2 1 0 m M K C I (vertical a r r o w s ) . F r a c t i o n s o f 0 . 8 5 m l w e r e c o l l e c t e d . T h e a c t i v i t y o f e a c h f r a c t i o n w a s m e a s u r e d in t h e p r e s e n c e o f a c o n s t a n t a m o u n t o f lentil r o o t R N A p o l y m e r a s e Ib. For f r a c t i o n s 1 - - 1 5 ( a d j u s t e d t o 7 0 m M KC1), 4 0 pl w e r e used; for the o t h e r s 1 3 . 3 pl.

(for 5 min at 60 ° C) before treatment (in these conditions temperature sensitivity of 5 results in a 20% decrease of activity only). A possible explanation for the stimulatory effect of 7 and 5 could be the presence of DNAase in the active fractions, thus producing nicks and free ends in the DNA- template in a non-specific way. This hypothesis cannot be retained because treatment of the D N A with either fraction 7 or 5 does not result in a significant increase of template activity when the D N A is re-isolated and used for R N A polymerase activity. These results rule out the possibility that the observed stimulation is due to a DNAase-like induced alteration of the template. The results clearly indicate that the active fractions ~/and 5 each contain a transcription factor that controls the activity of R N A polymerase Ib. Active fraction ~ is thermostable. After being heated for 5 rain at 90°C it loses only 10% of its stimulatory activity. On the other hand fraction 5 is thermolabiie; it loses all its activity when heated at 90°C for the same time. Transcription factor 5 can stimulate nucleolar polymerase Ib and the nucleoplasmic enzyme II equally well (Table IIb). On the other hand, factor "y is specific for the nucleolar enzyme, and has almost no stimulatory effect on the nucleoplasmic transcriptase.

397 T A B L E II S O M E P R O P E R T I E S O F T R A N S C R I P T I O N F A C T O R S ~" A N D 6 Unless o t h e r w i s e n o t e d t h e assays w e r e p e r f o r m e d u n d e r s t a n d a r d c o n d i t i o n s i.e, for 3 0 rain a t 3 5 ° C , w i t h lentil r o o t R N A p o l y m e r a s c Ib, n a t i v e calf t h y m u s D N A as t e m p l a t e a n d e i t h e r 4 0 pl s t a n d a r d b u f f e r ( a d j u s t e d t o 7 0 m M KC1), or f r a c t i o n 3' o r 5. E r r o r s given arc s t a n d a r d d e v i a t i o n s . D a t a r e p r e s e n t t h e a v e r a g e of five assays, e x c e p t f o r (c) ( w h e r e t h e y are t h e a v e r a g e o f t h r e e assays). ( a - - c ) F r a c t i o n 6 ( p r e v i o u s l y h e a t e d f o r 5 m i n a t 6 0 ° C ) o r f r a c t i o n 3, was t r e a t e d w i t h E n z i t e p r o t e a s e (Miles S e v a r a c ) 1 m g / m l f o r 3 0 rain at 3 7 ° C , f o l l o w e d b y r e m o v a l o f p r o t e a s e b y c c n t r i f u g a t i o n a t 47 0 0 0 × g for 3 0 rain. T h e s u p e r n a t a n t ( d i l u t e d 3-fold f o r f r a c t i o n 5) was t h e n a s s a y e d for s t i m u l a t o r y a c t i v i t y . C o n t r o l conr a i n e d s t a n d a r d b u f f e r 70 m M KCI. T h e d i g e s t i o n was p e r f o r m e d at 2 1 0 m M KC1 in t h e case of f r a c t i o n 5 to a v o i d b i n d i n g to c a r b o x y m e t h y l c e l l u l o s e ( t h e insoluble m a t r i x f o r E n z i t e p r o t e a s e ) . (d) R N A s s y n t h e sized in v i t r o w e r e e x t r a c t e d a n d p u r i f i e d as d e s c r i b e d in t h e t e c h n i c a l section. T h e a v e r a g e c h a i n l e n g t h L was c a l c u l a t e d a s s u m i n g t h a t t h e R N A s c o n t a i n e d 25% UMP, a n d t h a t i n i t i a t i o n s o c c u r only w i t h A T P a n d GTP. p m o l UMP L-

pmol ATP + pmol GTP

1 X-0.25

(e) Assays w e r e p e r f o r m e d , f o r t h e t i m e i n d i c a t e d , in a s t a n d a r d m e d i u m . R N A s w e r e c o l l e c t e d s i m u l t a n e o u s l y w i t h t w o t e c h n i q u e s , i.e. p r e c i p i t a t i o n w i t h t r i c h l o r o a c e t i c acid, a n d b i n d i n g to n i t r o c e l l u l o s e ( Millip ore filters). Fraction assayed None

( a ) P r o t e a s e s e n s i t i v i t y ( p m o l UMP i n c o r p o r a t e d ) With u n t r e a t e d f r a c t i o n 5.1 ± 0.4 With p r o t e a s e t r e a t e d f r a c t i o n 5.3 + 0.5 (b) A c t i v i t y ( p m o l UMP i n c o r p o r a t e d ) With R N A p o l y m e r a s e Ib 4.8 -+ 0 . 4 With R N A p o l y m e r a s e II 5.7 ± 0.5 ( c ) A c t i v i t y ( p m o l UMP i n c o r p o r a t e d ) w i t h d i f f e r e n t t e m p l a t e s With n a t i v e calf t h y m u s D N A 5.1 + 0.4 With d e n a t u r a t e d calf t h y m u s D N A 5.7 +- 0.4 With p o l y [ d ( A - T ) ] 7.2 ± 0.5 (d) C h a r a c t e r i s t i c o f R N A s s y n t h e s i z e d p m n l UMP i n c o r p o r a t e d 10.3 ± 0.6 pmol ATP incorporated 0 . 1 9 ± 0.01 pmol GTP incorporated 0.093 ± 0.005 A v e r a g e c h a i n l e n g t h ( n u m b e r of nucleosidcs) 146 +- 22 (e) Percentage of RNAs released during incubation A f t e r 15 r a i n o f r e a c t i o n 50 ± 5 A f t e r 3 0 rain o f r e a c t i o n 68 + 6

F r a c t i o n 3'

Fraction ( d i l u t e d 3-fold)

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± 0.7 2 0.5

15.9 7.5

± 1.2 -+ 0.5

10.4 7.2

± 0.8 -+ 0.6

18.0 18.5

-+ 1.2 ± 1.2

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± 0.7 -+ 0.5 -+ 1.4

15.6 12.6 38.2

-+ 1.1 ± 0.9 ± 2.5

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+ 5 -+ 5

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-+ 5 +- 7

The stimulatory effect of factors on the activity of enzyme Ib, when tested with different templates, is presented in Table IIc. Obviously, this effect is lower with denatured than with native calf thymus DNA. This result suggests that these factors play a role in the recognition of the double-stranded region of the DNA. The stimulatory effect is markedly pronounced when poly[d(A-T)] is used as template. Since its double helix can be easily opened, the above result suggests, but does n o t prove, that factors 7 and 5 favour the recognition of 'promotor-like' regions of the template by opening the double helix.

398

3. Nature of the step affected by factors ~ and 6 If these factors were initiation factors, one would expect a higher stimulatory effect at the start of the reaction, when the frequency of initiation events is high, than after a while, when a steady-state between initiation and elongation is reached. This is exactly what is obtained. The progress curve of UMP incorporation in vitro, in the absence of any factor, exhibits a lag which is nearly suppressed when either factor ~/ or factor 5 is present in the reaction medium, thus resulting in an efficient stimulation at the start of the reaction. These results are shown in Fig. 3. They suggest that factors 7 and 5 are initiation factors. The idea that factors 7 and 5 act on initiation events can be reinforced by analysis of the simultaneous incorporation of either [7.32p] ATP, or [ 7 - 3 2 p ] . GTP, and [ 3 H] UTP into the RNAs synthesized in vitro. It is well known that the nucleotide corresponding to the 5'-end of newly synthesized RNAs, generally a purine nucleotide [ 1 3 ] , is present as nucleoside triphosphate. Thus, it is easy to label selectively the 5'-end of RNAs with 7_32P.labelled purine nucleoside triphosphates, and to calculate the actual number of chains synthesized. When [ 3HI UTP is simultaneously incorporated inside the chains, as [ 3HI UMP, allowing an evaluation of the total number of nucleotides incorporated, it becomes possible to estimate the average chain length of the products. After extraction of these RNAs as described in the technical section, it can be shown (Table IId) that factors ~/ and 5 stimulate the incorporation of ATP in a more efficient way than that of UMP. The incorporation of GTP is not significantly affected when either factor "1, or 5 is present in the reaction mixture. On assuming that the nucleoside monophosphates are incorporated into the RNAs with the same frequency (25%}, it becomes possible to evaluate the average chain length of the transcription products obtained in the absence and in the

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Fig. 3. T i m e c o u r s e o f R N A s y n t h e s i s in t h e a b s e n c e ( o o ) , or in t h e p r e s e n c e o f f r a c t i o n 7 (o------@. p a n e l A ) o r ~, ( @ - - - - - - o , p a n e l B). T h e a c t i v i t y ratio ( i . . . . . . A) r e f l e c t s t h e relative s t i m u l a t i o n at t h e t i m e i n d i c a t e d .

399

presence of either factor 7 or 5. The results show that factors 7 and 6 stimulate initiations involving ATP, and seem to reduce the average chain length of the transcription products. The apparent decrease observed in the average chain length is n o t due to some nucleolytic activity contaminating the factors, for neither 3, nor ~ exhibited noticeable RNAase activity. An alternative explanation of the above results would be that the stimulatory fractions bring a b o u t an early release of R N A polymemse molecules and R N A chains, thus giving shorter chains and an enhancement of the rate of re-initiation. Therefore, an important point is to verify whether factors 7 and exert their action directly on initiation events rather than through a premature termination of R N A chains. It is well k n o w n that ternary complexes DNA • R N A polymerase • RNA, bind to nitrocellulose filters, allowing an estimation of the a m o u n t of R N A chains b o u n d to the template [ 1 4 ] . The percentage of chains released during incubations, in the absence and in the presence of stimulatory factors, can be calculated by comparison with the a m o u n t of radioactivity incorporated under standard conditions. It appears (Table IIe), that factors 7 and ~ do n o t affect the percentage of chains released during the reaction. Moreover, the activity of nucleolar e n z y m e Ib can be measured with and witho u t factors 7 or 5 in the presence of an increasing a m o u n t of spermidine. This polyamine stimulates transcription b y inducing the dissociation o f ternary complexes, leading to a rapid re-use of R N A polymerase molecules [15,16]. If fractions 7 and 5 were acting in this way, their stimulatory effect would be suppressed when assayed in the presence of spermidine. It is just the opposite which occurs (Fig. 4). The spermidine effect is weak when the reaction is catalyzed by the nucleolar e n z y m e Ib alone, and markedly stronger when factor 7 or 5 is present. These results clearly establish that factors 7 and ~ directly act on the initiation step rather than b y inducing a premature dissociation of

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[sp..m~.,] (~M) Fig. 4. E f f e c t o f s p e r m l d i n e c o n c e n t r a t i o n o n R N A p o l y m e r a s e I b - c a t a l y z e d R N A s y n t h e s i s . S t a n d a r d a s s a y s c o n t a i n e d v a r i o u s a m o u n t s o f s p e r m i d i n e in t h e a b s e n c e (o o) o r in the p r e s e n c e o f f r a c t i o n "/ (o-----o)

or6

(+ .....

-+).

400 the ternary complexes. The shortening of the R N A chains observed in the presence of stimulatory fractions could be induced by an 'overcrowding' of the template, consecutive to a higher frequency of productive initiation events. Discussion The experiments reported in this paper clearly show that various transcription factors can be isolated from the lentil roots. Two of them, 7 and 5, have been partly purified and characterized. These factors, when incubated with the nucleolar R N A polymerase Ib, lead to an increased transcriptional activity of the enzyme. Factors 7 and 5 only affect the initiation events, n o t t h e elongation steps. The level of factor 7 in the cell is highly controlled b y the auxin indole 3-acetate, whereas that of factor 5 is not. In the field of the m o d e of action of auxins at the molecular level, various interesting results have been reported over the past ten years. However, the results obtained by different research .groups have usually been discussed separately from those of others. It is thus of great importance to integrate these results, as well as those presented in this paper, into a unified scheme. It is the establishing of this scheme which is the central topic of this discussion. Various groups [3,4] have shown that auxin treatment of plant cells results in the loosening and the extension of the cell wall. This phenomenon, which occurs w i t h o u t any detectable lag [ 1 7 , 1 8 ] , can be mimicked by lowering the pH of the medium [19,20] and does n o t involve the synthesis of any new material, for cell elongation occurs even in the presence of cycloheximide [ 2 1 ] . These findings have been considered as indicative of an auxin-mediated activation of a plasma membrane-bound ATPase which initiates or stimulates a proton p u m p [ 2 2 ] , thus resulting in the weakening of cellulose-xyloglucan interactions in the cell wall [23]. Obviously, in addition to these early phenomena, auxin stimulates the de novo synthesis of proteins and of ribosomes [4,24]. However, it is well k n o w n that when the h o r m o n e is directly given to the isolated nuclei or to the chromatin, no stimulation of transcription is observed. The stimulation is detectable only when the auxin is given to the intact cell. This result clearly implies that, u p o n hormonal treatment, a signal is transported from the cytoplasm to the nucleus resulting in an increased transcription. One research group [25,26] has recently shown auxins to be tightly b o u n d to plasmalemma. In addition, vesicular fractions of the plasma membrane associated with auxin can stimulate the in vitro transcription of DNA by a crude R N A polymerase preparation [ 2 7 ] . The early effect of auxin on transcription, which can be detected in less than 1 h, involves an increased template availability of the chromatin [6]. It very probably necessitates the interaction of the DNA with a 'nuclear' protein, itself associated with the h o r m o n e [28]. This chromatin derepression leads to the synthesis of heterogeneous nuclear RNAs (HnRNAs) and short-lived messenger RNAs (mRNAs) [7]. These m R N A s would then be translated, in the cytoplasm, into specific proteins, one of them being factor 7. That factor would enter the nucleus, would associate with the nucleolar R N A polymerase Ib and would modulate its activity by giving to this enzyme the capability of recognizing new promoters on the DNA, leading to the massive synthesis of

401

new ribosomes [24] and to a general activation of protein biosynthesis. Some steps of this tentative process are still speculative. The nature o f the signal(s} which is transferred from the plasmalemma to the chromatin is unknown, as well as the mechanism of auxin-mediated derepression o f gene batteries. However, the protein factor 7, which plays a central part in this tentative process, is far more than a speculative entity. Its existence has been experimentally established, as well as its control by auxin. It is noteworthy that the mode of action of auxins, at the molecular level, seems strikingly similar to that of animal steroid hormones. For instance, factor 7 appears as a sort of counterpart in plants to the 'induced protein' of Gorski [ 2 9 ] , or to the 'Key Intermediary Protein' of Baulieu [ 3 0 ] . It could well be that the hormonal control of transcription in animals is very similar to that in plants. However, an obvious difference between the mode of action of auxins and that of animal hormones is that only in plants has an auxin-mediated mechanism evolved to allow the loosening and the extension of the cell wall. Acknowledgements This work was suppoi~ed by the C.N.R.S. (ATP Action des hormones). We are greatly indebted to Dr R. Rosset for numerous discussions and valuable comments. We would like to thank C. De Peretti and D. Veslin for excellent technical assistance. Thanks are due to Mrs Grossman for carefully reading the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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