Plant Science, 87 (1992) 19-27 19 Elsevier Scientific Publishers Ireland Ltd.

Development and line dependent variations of Petunia plasma membrane H+-ATPase sensitivity to auxin

J e a n - M a r i e F r a n g o i s a, A n d r 6 B e r v i l l 6 a a n d M i c h e l R o s s i g n o l b

aLaboratoire d'Am~lioration des Plantes, INRA, BV 21034 and bLaboratoire de Physiopathologie V~gktale, INRA. BVI540, 21034 Dijon Cedex (France)

(Received April 21st, 1992; revision received August I lth. 1992; accepted August 14th. 1992)

The response to auxin of the plasma membrane H+-ATPase from Petunia leaves was investigated in order (i) to check the general significance of sensitivity changes recently evidenced during development of tobacco and (ii) to test the intraspecific variability of the response to auxine. Two lines of Petunia hybrida, Tlvl and St40, were selected for their differential sensitivity to hormones in organogenesis experiments. Using a large variety of criteria, membrane fractions obtained were shown tO be greatly enriched in plasma membrane vesicles containing an active H+-ATPase able to generate a proton gradient. The proton translocation activity was stimulated by indoleacetic acid (IAA) on a biphasic way depending on the hormone concentration. In addition, the IAA concentration inducing the highest stimulation was found to change during plant development. A transient but dramatic decrease of the optimal auxin concentration (4 orders of magnitude, from 10 -5 M to 10 -9 M IAA) was observed at a time period which was assessed to cor- respond to floral induction. Both lines exhibited the same overall behavior towards auxin up to the end of floral induction. However, in the line Tlvl, the responsiveness to the hormone disappeared after floral induction, whereas a constant stimulation by 10 -5 M IAA was observed up to flowering in the line St40. These results are basically identical to those already obtained with tobacco. It is therefore concluded that a transient increase of the sensitivity to auxin of the plasma membrane H+-ATPase could be of general significance during floral induction, but in addition that some genetic variability could also be found between genotypes of a given species.

Key words: auxin; plasma membrane H÷-ATPase; proton translocation; line-dependent sensitivity; Petunia hybrida lines

Introduction

Auxins play a key role in many aspects of plant

development. Al though the mechanisms of act ion have not been elucidated yet, it seems likely that cytoplasmic as well as membrane located pr imary

target proteins are involved [1-3]. Regarding

Correspondence to: Michel Rossignol, Biochimie et Physiologie Vrgrtales, INRA, 34060 Montpellier Cedex I, France. Abbreviations: ACMA, 9-amino-6-chloro-3-methoxy-acridine: DTT, dithiotreitol; EDTA, ethylendiaminetetraacetic acid: EGTA, ethyleneglycol-bis-(anfinoethylether)N,N,N,N'-tetra- acetic acid; IAA, indoleacetic acid; Mes, 2-(N-morpholino)- ethanesulfonic acid; PEG, polyethylene glycol; PMSF, phenylmethansulfonyl fluoride; Tris, tris(hydroxy-methyl)- aminomethan.

0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Printed and Published in Ireland

membrane- located effects, it is well known that the plasma membrane H+-ATPase activity is modul- ated by auxins [4,5]. This auxin response has been recently demonstrated, in tobacco leaves, to be complex: depending on concentra t ion, auxin either stimulates, or inhibits, or does not affect pro ton translocat ion activity [6]. Moreover IAA concen- t rat ion inducing the highest H+-ATPase stimula- t ion is determined by plant age [6,7]. An impor tan t drop (3 orders of magni tude) of IAA concentra- t ion necessary to stimulate the H+-ATPase is observed dur ing plant development. This increase of I A A sensitivity has been correlated to the in- duct ion to flowering period [7]. Dur ing this period, changes of the H+-ATPase amoun t in plasma membrane have been observed. However, it was shown that var iat ions of plasma membrane

Ltd.

20

H÷-ATPase cannot completely explain the changes of IAA sensitivity observed [7]. Since IAA has no direct effect on H+-ATPase [8], one or several elements have to been involved in the reception and/or transduction of the auxin signal. In fact, evidence has been presented that several transducing systems well characterized in animals are also present in plants [9], but available data do not offer yet a clear rationale to analyze auxin signal [9]. Therefore other approaches such as the genetic approach should be promising [9]. Petunia constitutes a priori a material of choice in this view since (i) extensive genetic data are available [10] and (ii) differential responses to hormones be- tween lines are known [11] particularly concerning the sensitivity to auxin of the organogenesis from leave discs or protoplasts (H. Dulieu and J.P. Renaudin, pers. commun.).

The present work was undertaken with two lines of Petunia selected for a different responsiveness towards auxin in organogenesis experiments in order: (i) to determine whether or not the develop- ment dependent changes of IAA sensitivity of H ÷- ATPase from tobacco are of general significance and (ii) to investigate if different lines could reveal a variability of the plasma membrane H÷-ATPase sensitivity to auxin and offer a support for com- parative biochemistry studies or for a genetic ap- proach of the membrane response to auxin.

Material and Methods

Plant material The two lines of Petunia (Tlvl and St40) were

kindly provided by Dr. Cornu and Dr. Dulieu (INRA, Dijon, France). Petunia were sown and grown in a controlled environment chamber (20°C, 16 h light daily, 16 000 lux, 75% relative humidity). Four weeks after sowing, Petunia plants were planted in pots irrigated daily with a nutrient solution [6].

Membrane preparation Leaves were homogenized in 3 vol. (w/v) of 50

mM Tris-HCl buffer at pH 8.0 containing 0.5 M sucrose, 10 mM EDTA, 10 mM EGTA, 5 mM ascorbic acid, 5 mM DTT, 1 mM MgCI2 and 1 mM PMSF. The homogenate was filtered and cen-

trifuged at 16 000 × g for 20 min. The microsomal fraction was filtered on a 38-#m filter and cen- trifuged for 35 min at 50 000 × g. The pellet was resuspended in 5 mM KH2PO4/K2HPO4 (pH 7.8), 0.33 M sucrose and added to an aqueous PEG/dextran two-phase system to reach these final concentrations: PEG/dextran 6.75% (w/w) and 3 mM KC1. The two-phase system was vigorously shaken and the two phases were separated by gen- tle centrifugation. Upper and lower phases were washed twice with fresh corresponding phases. The upper phases were collected and added to 10 vol. of 10 mM Mes-Tris (pH 5.7), 0.33 M sucrose, 1 mM DTT, 1 mM PMSF. The membranes were pelleted (150 000 x g for 35 min) and resuspended in 10 mM Mes-Tris (pH 5.7), 20% glycerol, 0.25 M sorbitoi, 2 mM MgC12 and 1 mM PMSF.

Enzyme assays and proton translocation activity Enzyme assays were performed as previously

described [6,12]. Proton translocation activity was measured as in Refs. 6 and 7. by the initial fluorescence quenching rate of ACMA after ATP addition to membranes. Briefly, membranes (30 /~g) were diluted in 2 ml of 10 mM Mes-Tris buffer at pH 6.5 containing 50 mM KNO3, 50 mM KBr, 0.6 mM MgSO 4 and 2 #M ACMA. The sample was excited at 406 nm on a SLM 8000 spec- trofluorometer and ACMA fluorescence was recorded through a glass optical filter (50% T at 435 nm). For the study of auxin effect onto proton translocation, natives membranes were used within 2 h after preparation and only control measurements or auxin treatment giving the max- imal stimulation were repeated [6,7].

Statistical analysis The existence of sensitivity differences was

tested using the non-parametric test of Kruskal- Wallis [13] and the significance of sensitivity dif- ferences (i.e. the probability that the difference was true) was calculated by reference to the X 2 distribution [I 3].

Results

Membrane purification and character&ation The yield of the purification procedure, starting

21

from 100 g of fresh material of Tlvl Petunia line was 49.4 mg (S.D. = 23.5, n = 8) of protein in the microsomal fraction and 2.8 mg (S.D. = 0.9, n = 22) of protein in the final upper phase. Ap- proximately 6% of the microsomal fraction were therefore recovered. However, the ATP hydrolysis activity sensitive to vanadate was enhanced by about 330% in the latter fraction when compared to the former one. Similar yields were obtained for the line St40.

Figure 1 shows, for the line Tlvl, the effects of various treatments on the ATP hydrolysis activity of the membrane fraction present in the final upper phase. The activity was highly sensitive to vanadate and required Mg 2÷. Moreover, this membrane fraction was almost insensitive to the presence of oligomycin or azide (inhibitors of mitochondrial ATPase) or to nitrate (inhibitor of tonoplast ATPase). In the same way a very weak activity was detected when using IDP as substrate (marker for Golgi apparatus IDPase). Finally there was almost no phosphatase activity when

Control ~ - Magnesium ~ ,

+ Vanadate + Molybdate ~ I

+ Nitrate ~ / / A I Oligomyeine ~ ~ / / J ;

+ Azide ~ ~ ~ / / A I IDPase ~

PNPPase , , 0 50 100

Activity

Fig. 1. ATPase activities of the plasma membrane fraction from the Petunia line Tlvl. The ATPase activity was determin- ed, at 38°C, on 10/~g samples in 500 ttl of 50 mM Mes-Tris (pH 6.5), 100 mM KCI, 3 mM MgSO 4, 3 mM ATP and in the presence of lysophosphatidylcholine at a detergent/protein ratio of 6 (w/w). Activities are expressed as percentage of the control which amounted to 3.54/zmol of phosphorus liberated per min and per mg protein. Values are the means (±S.D.) of four sets of data. In the different conditions indicated, the medium was adjusted as it follows: - magnesium, no MgSO4; + Vanadate, + 0.25 mM vanadate; + Molybdate, + 1 mM molybdate; + Nitrate. + 100 mM KNO 3 instead of 100 mM KCI; + Oligomycine, + 5 mg • ml -t oligomycine; + Azide, + 5 mM NAN3; IDPase, + 3 mM IDP instead of 3 mM ATP; PNPPase, + 3 mM PNPP instead of 3 mM ATP.

using PNPP as substrate or molybdate as in- hibitor. In the presence of 3 mM M g - A T P the ac- tivity amounted to 3.54/zmol Pi liberated per mg plasmalemma protein and min (S .D.= 1.17, n = 7). The sidedness of the vesicles, determined using the ratio of ATPase activity in the absence of detergent over the ATPase activity in the presence of a 6 times excess of lysolecithin over proteins (w/w), indicated the presence of between 22% and 34% inside-out vesicles depending on the prepara- tion. Similar characteristics were observed with the line St40 towards markers, ATP hydrolysis activity and sidedness (not shown).

ATP hydrolysis activity was investigated as a function of the pH of medium (Fig. 2). Maximal activity was observed at pH = 6.5. The effect of several substrates (mono-, di- and tri-nucleotides) was also investigated (Fig. 3). The hydrolysis ac- tivity showed a very high selectivity for ATP. Finally, kinetic properties were determined con- cerning the sensitivity to vanadate and ATP con- centration. An inhibitor constant of 8.9/zM was found for vanadate (Fig. 4a). In the presence of an ATP regenerating system (see the legend of Fig. 4b), the ATP hydrolysis activity exhibited a plateau in the sub-millimolar range and fitting of the data according to Michaelis equation led to a K m value of 50/~M.

t~

5

4

3

2

1

0 5 6 7 8 9

pH

Fig. 2. pH effect on the ATPase activity of the plasma mem- brane fraction from the Petunia line Tlvl. ATP hydrolysis activity (#mol Pi • min -t • mg -I) was measured in various Mes-Tris buffers adjusted to pH-values between 5 and 9 with

either Mes or Tris.

22

ATP ITP . ~

CTP G T P UTP ADP IDP

AMP h PNPP , , ,

0 5 0 100 Activity

Fig. 3. Effect of various substrates on Tlvl plasma membrane fraction phosphohydrolase activity• Phosphohydrolase ac- tivities were assayed in standard conditions in the presence of 3 mM substrates. Activities are expressed as percentage of the ATPase activity, which amounted to 3.43 tcmol of phosphorus liberated per min and per mg protein and correspond to the average of two sets of data.

Proton translocation activity and auxin effect on the proton translocation activity

The H+-ATPase pro ton t ranslocat ion activity (Fig. 5) was measured in the presence of 250/zM ATP using the pro ton fluorescent probe A C M A [6,7]. The two lines of Petunia were found to ex-

hibit similar and relatively cons tant pro ton t ranslocat ion activities whatever the age of plants used for membrane prepara t ion (Fig. 6). Addi t ion of nigericin completely reversed the A C M A fluorescence to its initial value (Fig. 5a) showing that A C M A quenching was indeed due to pro ton accumulat ion inside the vesicles.

Addi t ion of IAA to native plasma membrane vesicles enhanced the pro ton t ranslocat ion activity of the H+-ATPase, as judged by the quenching of

A C M A fluorescence (Fig. 5b). The dose-response curve was biphasic (Fig. 7). U p o n addi t ion of auxin, the pro ton t ranslocat ion activity was first potentiated, but higher concentra t ions of auxin led to a disappearance of the effect. The concent ra t ion of IAA which was producing the max imum effect was called auxin sensitivity and the ampli tude of the auxin effect on pro ton t ranslocat ion activity was called auxin st imulation.

100

®

.-,~ so ° . ~ • " 0 "

• • . . , . • . o • •

0 t I ~ I n I

0 50 100 151

Vanadate (aM)

° ~

g

• o . . . . . . . . . • . . . . . . . . . O. . . , , ,

• O ' " "

8

0• i i I I i

0 200 400 600 800 1000

A TP (#M)

Fig. 4. Kinetic characterization of the TIvl plasma membrane H+-ATPase. (a) Inhibition of H+-ATPase by vanadate. Activ- ity was measured in standard buffer containing 9 mM MgSO 4 in order to eliminate surface electrostatic effects [18]. Results are expressed as percentage of inhibition of the control activity. Data points, from two sets of data, were fitted according to Michaelis equation (doted curve) where K i and maximal in- hibition were allowed to float. The best fit was obtained for K i = 8.9 #M. (b) Effect of ATP concentration on ATPase ac- tivity. Activity was measured in standard buffer and in the presence of an ATP regenerating system: 2 mM phosphoenol- pyruvic acid and pyruvate kinase (6 IU • ml-I). The best fit ac- cording to Michaelis equation (doted curve) was obtained for K m = 50 #M.

Effect of plant age on 1AA sensitivity of proton translocation activity

As it can be seen in Fig. 7, the auxin sensitivity was dependent on plant age (compare data for 43- day-old Petunia, full symbols, with data for 50-

23

A T P

, ' . . . Oo

. .

•o

II

"%. o - °

| ° ° o °

1 ~ n

® . . . . . • • .w• ~o%.d,

Nigericine •o

II

A T P ®

1 rain I

Fig. 5. Proton translocation activity of plasma membrane H+-ATPase from Petunia (a) Effect of nigericine (0.2 #g • ml -I) on pro- ton translocation of St40 line plasma membrane vesicles (30 #g protein). Reaction was started by addition of 250/zM ATP. Proton translocation activity, measured by the initial rate of fluorescence quenching amounted to 82 ± 3% fluorescence quenching per min -I • mg -]. (b) Auxin effect on proton translocation activity of Tlvl line plasma membrane vesicles (46-day-old plants, 30 ttg pro- tein). IAA was added from a 100-fold concentration solution in acetonitrile and control included 1% acetonitrile. Proton translocation activity amounted, for the control (light curve), to 92 ± 3% fluorescence quenching, min -l • mg -I, and to 122 ± 10% fluorescence quenching, rain - t • mg -l in the presence of 1 nM IAA (dark curve).

day-old plants, open symbols). This was also true for auxin stimulation which was low for the oldest plant (Fig. 7; see below). Therefore, these two parameters of auxin effect were systematically

150

1oo

~ 50

0

-.." ~ ................ •

I$ ..... O .... St40 ~- TIv1

I I I I I

40 50 60 70 80

Age of plants (days)

Fig. 6. Effect of plant age on the proton translocation activ- ity. Proton translocation activity was measured, in the absence of auxin, as described in Fig. 5a.

measured on plasma membrane vesicles of the two lines of Petunia ranging from 1 and 3 months of age (Fig. 8). The auxin sensitivity of the two lines of Petunia varied in a similar manner during plant development. For plants younger than 40 days the sensitivity was around 10 -5 M. In 6 days, between day 41 and day 46, the concentration of IAA need- ed to produce the maximum effect decreased dramatically to reach a minimum value (10 -9 M). In the next approx. 6 days, the sensitivity returned to its initial value (Fig. 7). Statistical analysis showed that all sensitivity changes reaching a fac- tor 10 were significant with a probability >95% (Fig. 9).

Auxin stimulation (height of the peak of auxin effect) varied differently for Tlvl and St40 during plant development. For Tlvl, auxin stimulation decreased starting from 46 days to disappear com- pletely around 55 days (Fig. 7a, insert). This behavior wasn't observed for St40 for which the auxin sensitivity remained constant thereafter (Fig. 7b, insert).

24

40

30

20

10

0,

I I I

- I 0

-20 , , i

lO "10 10"9 lO'S 10-7 10-6 lO'S 10-4 10-3

IAA (M)

Fig. 7. Auxin effect on the H+-ATPase-mediated proton translocation in plasma membrane vesicles from the Petunia line Tlvl. Plasma membrane vesicles were prepared from leaves of either 43-day-old (full symbols) or 50-day-old (empty symbols) Petunia. Pro- ton translocation in the presence of auxin is expressed as a percentage of the control activity in the absence of IAA. Activity of con- trols amounted, respectively to 82 4- 5% and 71 4- 8% fluorescence quenching, min -I • mg -l for 43-day-old and 50-day-old plants. For 43-day-old plants maximal stimulation (117 ~ 13% fluorescence quenching per min -l mg -I) occurred in the presence of 2/~M IAA and for 50-day-old plants in the presence of 0.2 #M IAA (85 4- 2 fluorescence quenching per min -I • mg-l).

Discussion

As the present work constituted the first attempt to purify plasma membrane vesicles from Petunia, effort was devoted to characterize the membrane fractions obtained. All our data indicate that the phase partitioning procedure allows one to prepare a membrane fraction which contains a very active H+-ATPase exhibiting characteristics usually found for the plasma membrane H +- ATPase [14,15]. In addition, almost no con- taminating activity was detected, so it can be assumed that the fractions used here were highly enriched in plasma membrane.

Proton translocation by the H÷-ATPase of the two Petunia lines appears to be sensitive to the ad- dition of auxin. In addition, the response is biphasic with respect to the hormone concentra- tion. Comparable data are available only on tobacco where similar results were obtained with normal [6,7] or transformed [16] plants. Taken together, these d a t a suggest t h a t this kind of

response could correspond to a characteristics of the reception and transduction pathway which lead to ATPase stimulation in plant plasma mem- brane. On the other hand auxin sensitivity of Petunia H+-ATPase changes during plant development: it undergoes a transient but dramatic increase in sensitivity (approx. 4 orders of magnitude) between days 40 and 52. Similar patterns have been also observed with tobacco [6,7,16]. On tobacco, the transient sensitivity in- crease occurs roughly at the induction to flowering period [7]. No direct determination was made here of the period where induction to flowering occurs on Petunia. The 40-52-day period is concomitant, however, with morphological changes of plants, such as internode elongation, which are known take place after floral evocation [16]. On this basis it could be hypothetized that the increase of the membrane sensitivity to auxin occured at the time period of floral induction. Taken together the present results suggest that the shape of dose- response curves as well as the occurence of a

25

<

1 0 -s

1 0 -6

10 -7

1 0 -s

I 0 -9

10 -s

1 0 -6

10 -7

1 0 -s

1 0 -9

® i

® I

I i

30 50 70

d a y s I i

I I i

9 0

30 50

• • ! u 7 1 g ' ' 'o 50 9 0

d a y s

I

70 90

D a y s

Fig. 8. Effect of plant age on IAA sensitivity and stimulation by IAA of the proton translocation activity of Petunia plasma mem- brane H+-ATPase. Auxin sensitivity was measured, on Tlvl line (a) or St40 line (b) by the auxin concentration which induced the highest stimulation of the proton translocation activity. Data are from 3 complete experiments for St40 and 4 for Tlvl with approx. 300 Petunia plants for each type of plant material. For plants of the same age, the sensitivity differences between experiments did not exceed a factor 5. Inserts indicate the corresponding stimulations (percentage over control proton translocation activityl.

changing sensitivity during floral induction, previously observed only on tobacco, could be o f general significance in plants.

Al though both lines o f Petunia studied exhibit the same overall behavior towards auxin, one striking difference concerns the disappearance o f the response on the Tlvl line after floral induction. This apparent ly does not involve directly the H ÷-

ATPase himself, since the p ro ton translocation ac- tivity remains almost constant up to approx. 80 days (Fig. 6). One possible cause could concern changes o f the membrane permeability or vesicles sealing. Al though this cause was not investigated here, the fact that the p ro ton translocation activity remains constant argues against this hypothesis as long as the number o f ATPase molecules per unit

26

e ~

100

80

60

40

20

0

0¢

I i i

++OOe09 O~ ++0 +

0 0

O

I I I

10 t 10 z 10 3

Sensitivity ratio

1 0 4

Fig. 9. Probability of a significant difference between the sen- sitivities to auxin of two plasma membrane preparations accor- ding to the ratio of their sensitivities. Proton translocation activities in the presence of auxin were systematically analyzed using the Kruskal-Wallis test for plants ranging between 43 and 53 days. The probability of rejection of the null hypothesis, for each of the 30 pairs of data which were compared, was plotted versus the ratio of the sensitivities measured for the two members of the pair.

membrane can be considered also to be constant. An other possible interpretation is that some ele- ment of the reception and transduction pathway becomes deficient after floral induction on the line Tlvl. In this case however, such modifications should not affect further plant development since both lines flower at the same age. Interestingly, on the other hand, leaf explants from the two lines ex- hibit different competence to organogenesis [11; H. Dulieu, pers. commun.]. At the present time, no clear relationship can be derived from the two kinds of data, but these results suggest that mem- brane steps could be possibly involved in the in vitro organogenesis response to hormones.

In conclusion, data presented here corroborate results obtained until now only with tobacco on the occurence of a changing membrane sensitivity to auxin during plant development. In addition they indicate that this changing sensitivity is susceptible to genetic variability. In this view, the extensive genetic background available on Petunia could offer an opportunity for future genetic analysis of both membrane response to auxin and in vitro competence to organogenesis.

Acknowledgments

This work was supported by a CEC fellowship to J.M.F.

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