Planta

Planta (1986)167:129-132

9 Springer-Verlag 1986

Incubation of corn coleoptiles with auxin enhances in-vitro fusicoccin binding P. Aducci, A. Ballio and M. Marra Gruppo di Chimica Biologica e Strutturistica Chimica, Facolt~ di Scienze, Universitg di Roma " L a Sapienza", Cittg Universitaria, 1-00185 Rome, Italy

Abstract. Binding of fusicoccin (FC) to microsomal preparations of corn (Zea mays L.) coleoptiles is enhanced after incubation of the tissue with indole3-acetic acid (IAA). Treatment of the kinetic data according to Scatchard shows that the enhancement is a consequence of an increase in the number of high-affinity FC-binding sites without changes of their KD. The minimal effective concentration of IAA is 10 -7 M; above 1 0 - 5 M the effect declines. The stimulation is insensitive to protein-synthesis inhibitors (cycloheximide and puromycin). The same effect is observed with the synthetic auxins 2,4-dichlorophenoxyacetic acid and naphtaleneq-acetic acid while it is abolished by the auxin antagonists naphtalene-2-acetic acid and p-chlorophenoxyisobutyric acid. Since the above effect is only observed with intact tissue and not after incubation of IAA with microsomal preparations, a direct interaction of IAA with the FC-binding sites is ruled out and an alternative mechanism must be sought.

Key words: Auxin and fusicoccin - Fusicoccin Zea (fusicoccin binding).

Introduction

Similarities and differences in the effects produced on plant growth by auxin and fusicoccin (FC) have been extensively discussed (Lado et al. 1972; Cleland 1976; Marr6 1979; Rubinstein and Cleland 1981; Kutschera and Schopfer 1985a, b). There is general agreement that their modes of action Abbreviations: 2,4-D=2,4-dichlorophenoxyacetic acid; F C = fusicoccin; [3H]FC=3H-labeled dihydrofusicoccin; I A A = i n dole-3-acetic acid; 1-NAA = naphtalene-l-acetic acid; 2-NAA=naphtalene-2-acetic acid; PCIB=p-chlorophenoxyisobutyrie acid

are not identical and most relevant in this respect is the observation that they bind in vitro to different sites (Dohrmann et al. 1977; Ballio et al. 1981). In the present paper we report data indicating that when indole-3-acetic acid (IAA) is supplied to segments of corn coleoptiles, more FC is bound in vitro. This is the first report showing that among the informations carried by IAA, one is decoded at the primary site of action of FC. Some preliminary observations of this effect have been reported previously (Aducci et al. 1979).

Material and methods Corn (Zea mays L., cv. XL 342, purchased from Dekalb, Mestre, Italy) caryopses were sown on tap-water-moistened vermiculite and kept 4 d at 29 ~ C in the dark. Coleoptile segments were dissected in ordinary light, incubated in the presence and the absence of IAA or other substance, rinsed with glass-distilled water, and immediately homogenized to get the microsomal preparations. These were prepared according to Dohrmann et al. (1977). Binding of FC was determined as described by Aducci et al. (1984a). The particulate preparations used for FC-binding were also used for the assay of IAA-binding (Jacobs and Hertel 1978) which was performed with I 0 - 7 M [14C]IAA. In order to compare the FC-binding of microsomal preparations obtained from tissue incubated in the absence of other substances (control) with that of preparations obtained from tissue incubated with added IAA or other auxins, the binding data are reported as concentration of bound ligand per mg of particulate protein. Protein concentration was determined with a modified Lowry procedure (Lees and Paxman 1972). Scatchard analysis was according to a previous paper (Aducci et al. 1984a). Corn coleoptiles (2 g) were incubated at 26 ~ C under gentle shaking in 50 ml citrate buffer (pH 5.5) containing 2.5 mM sodium citrate, 1.25 m M MgSO4, 10 m M KC1. When present, IAA and auxin analogs were at a 10 S M concentration. In the experiments with protein-synthesis inhibitors, the tissue was preincubated 1 h with 3-10 -5 M cycloheximide or with 10 - 4 M puromycin, then rinsed with glass-distilled water and incubated with IAA under the above reported conditions. In each experiment the physiological activity of auxin was controlled by an independent measurement of coleoptile elongation.

130 Fusicoccin was prepared according to Ballio et al. (1968); [3H]FC was prepared by catalytic tritiation of FC (Ballio et al. 1980); its specific activity was 3.45.10 9 Bq.mg -1. Indole-3acetic acid was purchased from Polysciences Inc., Warrington, Penn., USA, [I-14C]IAA(I.20 - 107 Bq.mg 1) was a gift of Professor R. Hertel (Freiburg, FRG) who also generously provided 1-IAA, naphtalene-2-acetic acid (2-NAA) and p-chlorophenoxyisobutyric acid (PCIB). Cycloheximide and puromycin were purchased from Sigma, St. Louis, Mo., USA. Other chemicals were purchased from Merck, Darmstadt, FRG.

P. Aducci et al. : Enhancement of fusicoccin binding by auxin 100 r

._o

_~ 50

0

Iog [IAA] (M) Results

Microsomal preparations of corn coleoptiles obtained from tissue which had been incubated in the presence of IAA bound more FC than control preparations made from tissue which had not come in contact with the auxin. On averaging the results of twenty experiments run under identical conditions, a 50% stimulation was calculated, with data ranging from 30 to 100%. Attempts to assess the reasons for this variation were unsuccessful. In some experiments the IAA effect on FCbinding was already detected after 10 min incubation of the tissue with the auxin, a result which was not always reproducible. Therefore, the length of the incubation was standardized at 90 min, when saturation had always been achieved. The IAA stimulation was concentration-dependent (Fig. 1). It reached a maximum in the interval 1 0 - 6 - 1 0 - 5 M and smoothly declined at higher concentrations. Treatment of coleoptiles with two synthetic auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphtalene-l-acetic acid (I-NAA), was as effective as with I A A (Fig. 2). The effects on growth of the natural and the two synthetic auxins were checked by measuring the elongation responses of multiple samples of coleoptile segments after 24 h exposure to the same solutions used for the FCbinding enhancement tests. An approx. 40% stimulation of elongation was observed for each of the three auxins. Next, two auxin antagonists, PCIB and 2-NAA, were examined. When these compounds (10 -4 M) were added to the IAA-containing medium in which coleoptiles were incubated, growth was inhibited and the stimulating effect of IAA on FC-binding to the microsomal membranes was completely abolished (Fig. 2). In order to establish whether the effect of auxins on FC-binding was dependent on protein synthesis, puromycin and cycloheximide were added to the tissue 1 h before starting the incubation with IAA. As expected, the two substances impaired the elongation response of coleoptile segments to IAA, but were unable to affect the stimulation of

Fig. 1. Dependence of IAA stimulation of FC-binding on auxin concentration

IAA

'I-NAA

2o4-D

IAA + 2-NAA

50

IAA + PCIB

c-

._o :3

25

[

I i

I

Fig. 2. Stimulation of in-vitro FC-binding after incubation (90 min) of corn coleoptiles segments with IAA, two synthetic auxins (2,4-D and 1-NAA) and mixtures of IAA and two auxin antagonists (PCIB and 2-NAA) Table 1. Effect of cycloheximide (3.10 -5 M) and puromycin (10 -4 M) on IAA (10-5 M) stimulation of FC-binding

Stimulation (%)

Exp. 1 Exp. 2

IAA

Cycloheximide

Puromycin

IAA + Cycloheximide

IAA + Puromycin

+60 +45

0 -

+10

+65 -

+40

FC-binding induced by auxin (Table 1). The FCbinding of microsomal preparations from coleoptiles incubated without auxin was also insensitive to the presence of the protein-synthesis inhibitors during incubation. The data of [3H]FC-binding to corn-coleoptile membranes prepared from control tissue and from tissue incubated with IAA were analysed according to Scatchard (1949), using a non-linear leastsquares fitting program (Aducci et al. 1984a). The data fit into theoretical curves representative of the binding behaviour for two classes of specific sites in each sample (Fig. 3). The auxin had no significant effect on the two dissociation constants

P. Aducci et al. : Enhancement of fusicoccin binding by auxin

0.30 [k]

@

B

[L]F 0.20

0.10

m

0.0 -9

-8

-7

- - I

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tog [ L] tM)

F Fig. 3. Analysis of binding data according to Aducci et al. (1984a). Continuous lines correspond to the best fitting for two classes of sites. Data are shown from three separate sets of experiments run with microsomal preparations obtained from segments of corn coleoptiles incubated in the presence (e) and in absence (a) of IAA

(KD) which have values coincident with those reported previously (Aducci et al. 1984a) for the binding of FC to microsomal preparations prepared under standard conditions. However, after auxin treatment of the tissue, the number of highaffinity binding sites (nl) increased by 40% [(2.6_+0.10)" 10 -13 m o l ' g -1 fresh weight, as compared with (1.4_+0.09).10 -13 mol-g -1 fresh weight for the control not preincubated with IAA]. It was difficult to establish an effect on the number of low-affinity sites (n2), since the determination of this absolute value is subject to large uncertainty. The stimulation of FC-binding observed when IAA is added to an in-vivo system is not duplicated in an in-vitro system. No effect was ever detected when microsomal fractions of corn coleoptiles were incubated for different lengths of time (up to 1 h) with different amounts (from 5-10-6 up to 10 4 M) of IAA before addition of [aH]FC (data not shown). It was verified that the same fractions retained the ability to bind [14C]IAA; FC did not compete in this assay. Discussion

The present paper reports on the enhanced ability of corn-coleoptile microsomal preparations to bind FC in vitro when the tissue has been previously incubated with either IAA, 2,4-D, or 1-NAA. This effect is concentration-dependent, is abolished by

131

auxin antagonists (2-NAA and PCIB), is independent of protein synthesis and requires the integrity of the tissue. In fact, the FC-binding stimulation is not observed when microsomal fractions are incubated with IAA, and thus cannot depend upon a direct interaction of IAA with the FC-binding sites. This is in agreement with previous observations showing that auxin and FC bind to different sites (Dohrmann et al. 1977; Ballio etal. 1981). Consequently, the mechanism which leads to the stimulation of FC-binding by auxin must involve the mediation of some as-yet-unidentified secondary messenger present in the tissue. Scatchard analysis indicates that the incubation with IAA increases the number of FC-binding sites. In recent years, two independent findings, which are probably pertinent to the regulation of these sites, have been reported. First, evidence for the occurrence in plant tissues of compounds capable of interacting in vitro with FC-binding sites and of inhibiting FC-stimulated H§ has been yielded (Aducci et al. 1980). Second, it has been shown that the FC-binding sites are phosphorylated glycoproteins easily inactivated by endogenous phosphatases and-or c~-mannosidases (Aducci et al. 1984b). In the light of this information, one can postulate that an increase in the number of FCbinding sites might arise either by displacement of the physiological ligands ('unmasking'), or by phosphorylation and-or mannosylation of sites which had been made inactive by some endogenous hydrolase ('reactivation'). Attempts to demonstrate the release of specific endogenous ligands during incubation of corn tissue with IAA have failed so far (data not shown). The stimulation by IAA of reactions capable of bringing inactivated binding sites back to their original function is particularly attractive. In this context the specific promotion by IAA and 2,4-D of calcium release from soybean microsomal preparations (Buckhout et al. 1981) is relevant, since an increase in free calcium ions is the prerequisite for the activation of a number of enzymes, among them some protein kinases, through the formation of a complex with calmodulin (Marm~ and Dieter 1983). Also, an increase in cytoplasmic calcium ions is possibly the cause of an increased vesicle flow from the Golgi apparatus to the cell surface (Hertel 1983), with the likely consequence that this intracellular traffic promotes an increased concentration of some plasma-membrane glycoproteins. We are grateful to Dr. M. Coletta for help in the computer analysis of binding data and to Drs. R. Federico and D. Scalorbi who carried out some preliminary experiments. Fruitful discussions with Professor R. Hertel are acknowledged. This

132 research was supported in part by the Italian Ministry of Education and in part by the Italian Research Council (C.N.R.). It was in partial fulfilment of the requirements for M. Marra's Ph.D. in Biochemistry (University of Rome "La Sapienza", Rome, Italy).

References Aducci, P., Ballio, A., Federico, R., Scalorbi, D. (1979) Enhancement of fusicoccin binding to corn coleoptiles plasma membranes by in vivo treatment with indole-3-acetic acid. G. Bot. ltal. 113, 191 Aducci, P., Ballio, A., Fiorucci, L., Simonetti, E. (1984b) Inactivation of solubilised fusicoccin-binding sites by endogenous plant hydrolases. Ptanta 160, 422-427 Aducci, P., Coletta, M., Marra, M. (1984a) An improved Scatchard analysis of fusicoccin-binding to maize coleoptile membranes. Plant Sci. Lett. 33, 187-193 Aducci, P., Crosetti, G., Federico, R., Ballio, A. (1980) Fusicoccin receptors. Evidence for endogenous ligand. Planta 148, 208-210 Ballio, A., Carilli, A., Santurbano, B., Tuttobello, L. (1968) Produzione di fusicoccina in scala pilota. Ann. Ist. Sup. Sanitfi 4, 317-322 Ballio, A., Federico, R., Pessi, A., Scalorbi, D. (1980) Fusicoccin binding sites in subcellular preparations of spinach leaves. Plant Sci. Lett. 18, 39-44 Ballio, A., Federico, R., Scalorbi, D. (1981) Fusicoccin structure-activity relationships: in vitro binding to microsomal preparations of maize coleoptiles. Physiol. Plant. 52, 476-481 Buckhout, T.J., Young, K.A., Low, P.S., Morrfi, D.J. (1981) In vitro promotion by auxins of divalent ion release from soybean membranes. Plant Physiol. 68, 512-515 Cleland, R.E. (1976) Fusicoccin induced growth and hydrogen

P. Aducci et al. : Enhancement of fusicoccin binding by auxin ion excretion of Arena coleoptiles: relation to auxin respones. Planta 128, 201-206 Dohrmann, U., Pesci, P., Cocucci, S.M., Marr+, E. (1977) Stimulating effect of fusicoccin on K +-activated ATPase in plasmalemma preparations from higher plant tissues. Plant Sci. Lett. 8, 91-98 Hertel, R. (1983) The mechanism of auxin transport as a model for auxin action. Z. Pflanzenphysiol. 112, 53-67 Jacobs, M., Hertel, R. (1978) Auxin binding to subcellular fractions from Cucurbita hypocotyls: in vitro evidence for an auxin transport carrier. Planta 142, 1-10 Kutschera, U., Schopfer, P. (1985a) Evidence against the acidgrowth theory of auxin action. Planta 163, 483-493 Kutschera, U., Schopfer, P. (1985b) Evidence for the acidgrowth theory of fusicoccin action. Planta 163, 494-499 Lado, P., Pennacchioni, A., Rasi Caldogno, F., Russi, S., Silano, V. (1972) Comparison between some effects of fusicoccin and indole-3-acetic acid on cell enlargement in various plant materials. Physiol. Plant Pathol. 2, 75-85 Lees, M.B., Paxman, S. (1972) Modification of the Lowry procedure for the analysis of proteolipid protein. Anal. Biochem. 47, 184-192 Marm6, D., Dieter, P. (1983) The role of Ca 2§ and calmodulin in plants. In: Calcium and cell function, vol. 4, pp. 263-311, Cheung, W.Y., ed. Academic Press, New York London Marr6, E. (1979) Fusicoccin, a tool in plant physiology. Annu. Rev. Plant Physiol. 30, 273-288 Rubinstein, B., CMand, R.E. (1981) Responses of Arena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic acid. Plant Physiol. 68, 543-547 Scatchard, G. (1949) The attractions of proteins for small molecules. Ann. N.Y. Acad. Sci. 51,660-672

Received 24 July; accepted 4 October 1985