Planta (198l, 151:434-438

P l a n t a 9 Springer-Verlag 1981

Auxin-Stimulated ATPase in Membrane Fractions from Pumpkin Hypocotyls (Cucurbita maxima L.) Gtinther F.E. Scherer Botanisches Institut, UniversitS.t Bonn, Venusbergweg 22, D-5300 Bonn 1, Federal Republic of Germany

Abstract. Membrane fractions from C u c u r b i t a m a x i m a hypocotyls were isolated in a medium which inhibits the action of endogenous phospholipases. After removal of soluble phosphatases by Sepharose 2B-CL column chromatography, an auxin-stimulated ATPase activity was found in membrane fractions from linear sucrose gradients. In the presence of 10-4 M phenylacetic acid (PAA), the stimulation by indol-3-acetic acid (IAA) exhibited a bimodal concentration dependence with maximal stimulation of about 50% at 10 - 6 M IAA. Without PAA, only a high concentration of 10-4M IAA was stimulatory, whereas 10-6M IAA had no apparent effect and 10-8M IAA exhibited weak inhibition. PAA alone had only weak or no effects. The effects of IAA must be considered as hormone-specific. The ATPase activity in the presence of 10-4 M PAA was activated only by 2,4dichlorophenoxyacetic acid (2,4-D), an active auxin analogue, but not by the inactive stereoisomers, 2,3-D and 3,5-D. Comparison with marker enzyme profiles suggested that part of the auxin-stimulated ATPase was localized on plasma membranes as well as other compartments. Thus, the auxin-stimulated ATPase may become a useful tool in the investigation of the mechanism of action of auxin. Key words: ATPase - Auxin - Membrane fractionation

Cueurbita

-

Hypocotyl

Introduction

Ten years have elapsed since the acid growth hypothesis was formulated to explain the mechanism of auxinstimulated growth (Cleland 1971 and 1973; Hager Abbrevations: 2,4-D = 2,4-dichlorophenoxyacetic acid; 2,3-D = 2,3-

dichlorophenoxyacetic acid; 3,5-D=3,5-dichlorophenoxyacetic acid; IAA=indol-3-acetic acid; PAA=phenylacetic acid; M E S = (2-(N-morpholino))-ethanesulfonic acid; EDTA=ethylenediamine tetraacetic acid

0032-0935/81/0151/0434/$01.00

et al. 1971). Studies on the hormone-stimulated proton transport into the cell wall and the surrounding media (e.g., Cleland 1973 and 1975; Cleland and Lomax 1977; Lado et al. 1976; Colombo et al. 1979) and on the auxin-regulated ion transport (e.g. Marr~ et al. 1975; Lado et al. 1976; Cleland et al. 1977; Colombo et al. 1979) suggested that regulation of ion transport across the plasma membrane may be a significant event in the auxin-stimulated cell growth. Thus, biochemical research into the mechanism of auxin-stimulated growth has centered around auxinmembrane interaction. An approach toward an understanding of the action of auxin on membranes has been a search for an auxin-regulated, membrane-bound enzyme. In principle, a hormone-sensitive, membrane-bound ATPase is a likely candidate for the regulation of proton flow across the plasma membrane. A report of an auxin-stimulated, plasma membrane-bound ATPase by Kasamo and Yamaki (1974) was challenged by Cleland and Lomax (1977) and Venis (1977). However, we demonstrated effects of 2,4-D, an artificial auxin, on two or possibly three phosphatases in membrane preparations from soybean hypocotyls (Scherer and Morr6 1978 a). One of these phosphatases was an ATPase. More recently, similar results have been reported with rice root microsomes (Erdei et al. 1979). Here, we present additional results on an auxinstimulated ATPase in membrane fractions from pumpkin hypocotyls. These results may offer a first link between auxin binding (Venis 1977; Hertel 1979), on the one hand, and on rapid ion transport processes influenced by auxin in vivo on the other.

Materials and Methods Plant material. Pumpkin seeds ("Gelber Zentner") were sterilized

for 5 rain with a 0.3% sodium hypochlorite solution and grown at 25 ~ C for 6 days in the dark on moist cotton.

G.F.E. Scherer: Auxin-stimulated ATPase

435

Chemicals9 Nupercaine was a generous gift from CIBA-GEIGY (D-7867 Wehr, FRG) and 3,5-D was kindly given by Dr. N. Amrhein (Bochum, FRG). All other chemicals were from commerciM sources of the highest purity available9 Membrane Isolation. Pumpkin hypocotyls (45 g) were chopped finely in ice-cold isolation buffer (1:1 u/v) of 8% choline chloride (w/v), 8% ethanolamine (v/v), 10 m M dithioerythritol, 20 mM EDTA, 0.3 mM nupercaine, and 10 mM sodium glycerol-l-phosphate titrated with HC1 to pH 7.5. Phenylmethylsulfonylfluoride (2 mM) was added as a protease inhibitor prior to homogenization. The pieces were ground in an all-glass homogenizer of the PotterElvejehm type cooled with an ice water jacket. Subsequently, the temperature was kept at 4 ~ C. The homogenate was filtered through a nylon cloth and centrifuged at 3,000 g for 10 min. The supernatant was chromatographed on a Sepharose 2B-CL column (3.30cm; flow rate 70 ml h -1) in 4% choline chloride (w/v), 4% ethanolamine (v/v), 2 m M dithioerythritol, 20 mM EDTA, and 0.15 mM nupercaine, pH 7.5 ( = c o l u m n buffer)9 Membrane-containing fractions were identified by their turbidity, pooled, and layered onto a sucrose gradient consisting of 0.3 ml 2 M and 1.6 M sucrose; 1 ml of 1.4 M, 1.2 M, t.1 M, 1.0 M, 0.9 M, 0.8 M, 0.7 M sucrose;and 0.5 ml of 0.6 M, 0.45 M and 0.3 M sucrose9 Gradients were poured 24 h prior to use and stored at 4 ~ C until needed9 All sucrose solutions were prepared in column buffer9 Gradients were spun for 4 6 h at 27,000 rev rain 1 (Spinco SW 27 rotor with slim buckets) and then fractionated. The protein content of the supernatant was routinely checked and was always very low. Pooled gradient fractions from parallel gradients were chromatographed on Sephadex G 50 columns (1.30 cm) and equilibrated into 10 m M MES/NaOH (pH 6,5). Protein recovery was 40-60%. ATPase assays were done immediately after the Sephadex chromatography and all other marker enzyme activities subsequently tested9 Enzyme Assays. The ATPase assay was adapted from Hodges and Leonard (1972). Enzyme (200 gl) was added to a mix of 50 pl of concentrated incubation medium containing hormone, 15 mM Na2ATP, 7.5 mM MgCI2, and 0.11 M MES/NaOH (pH 5.5). Activity was assayed for 30-60 min at 18 ~ C. The reaction was stopped by 0.8 ml cold 3.5% perchloric acid and the tubes kept on ice for 2 h. Then, 60 gl 45% KOH was added which nearly neutralized the assay and inorganic phosphate assayed by the method outlined by Scherer and Morr4 (1978). Glucan synthetases were assayed by the procedure by Jesaitis et al. (1977). For glucan synthetase I the incubation time was 15 min and for glucan synthetase II 60 min. Cytochrome c reductase and oxidase were assayed as described by Hodges and Leonard (1972). Cytochrome c reductase was measured in the presence o f 0.022 mg ml- ~ antimycin A and i m M

KCN, and the cytochrome c oxidase in the presence of 0.1% Triton X 100 (Jesaitis et aI. I977). Inosine diphosphatase was assayed by the method of Bowles and Kauss (1976). The assay was scaled down to a total volume of 0.2 ml and stopped after I h incubation at room temperature. ~-Mannosidase was assayed by a modified Boller and Kende (1979) method 9 To 0.4 ml 0.1 M succinate/NaOH buffer (pH 5.5) containing 3.7 mM p-nitrophenyl-c~-D-mannoside, 0.1 ml enzyme was added and incubated for 5 h at 35 ~ C. The reaction was terminated with 1 ml 1 M Na2CO3 and the absorbance read at 405 rim. Acid phosphatase was assayed in a similar manner to c~-mannosidase (Boller and Kende 1979) using p-nitropbenyI-phosphate as a substrate. Incubation time was l h at room temperature with a suitably diluted enzyme. Carotinoids. Total lipids were extracted according to Bligh and Dyer (1959) and the chloroform phase was adjusted with methanol to the 1.5 fold of the original water volume. The absorbance density was read at 445 rim. Protein was determined with the method of Lowry et al. (1951) after precipitation with 10% trichloroacetic acid. Lipid-bound inorganic phosphate. Lipids were extracted with the method of Bligh and Dyer (t959) and digested, and phosphate was determined according to Rouser et al. (1966)9

Results and Discussion

Chromatography of postmitochondrial supernatant on Sepharose 2B-CL effectively separated membranebound enzymes (e.g., cytochrome c oxidase and reductase) from mainly soluble enzyme activities (Fig. 1). Membrane-containing fractions chromatographing in the void volume were pooled and centrifuged on linear sucrose gradients. After centrifugation and Sephadex chromatography, gradient fractions were assayed for the presence of an auxin-stimulated ATPase activity. Auxin alone stimulated ATPase activity in gradient fractions only at a hormone concentration of 10-4M (Fig. 2a). With 10 -8 M IAA a weak inhibition was observed, whereas 10-6 M IAA had no apparent effect. Typically, this inhibitory effect was most prominent in fractions with the highest activity of NADH/cytochrome c reductase (Antimycin A-insensitive), a marker for ER (compare Fig. 2a with 2c). This inhibition of ATPase activity was abolished by the addition

2.0 [3 o.

\

E

1.5-

~E

~ ,01-

o o~

g~

wu -g-g

uL~

5

froction

10

froction

of 10 -4 M PAA (Fig. 2b). PAA inhibits auxin binding to ER (Jacobs and Hertel 1978; Dohrmann et al. 1978). Whether the PAA effect on auxin-stimulatable ATPase is related to the PAA effect on auxin binding to ER is not known. Interestingly, in the presence of 10 . 4 M PAA, ATPase activity was most strongly stimulated by 10 -6 M IAA. The stimulation ranged from 35% to 50%, or more in individual fractions (Fig. 2b and 3). Tile effects of PAA alone were always considerably smaller than auxin effects and thus the stimulation of ATPase activity must be considered as auxin specific. The hormone specificity was further investigated by comparing the effects of one active and two weak or inactive auxins. Together with PAA, only the active auxin analogue 2,4-D stimulated ATPase activity in gradient fractions, but the stereoisomers 2,3-D or 3,5D did not (Table 1). In an attempt to localize the auxin-stimulated ATPase activity with a specific membrane class, a comparison of marker enzymes was made (Fig. 2 and Fig. 3). This comparison revealed that auxin-stimulated ATPase was found in dense fractions where only glucan synthetase II, a plasma membrane marker (Quail 1979), and cytochrome c oxidase, a mitochondrial marker, occurred. In these dense fractions the hormone-sensitive component was likely to be located

Fig. 2a-d. Stimulation of ATPase activity by auxin. A postmitochondrial supernatant was freed from soluble protein by column chromatography on Sepharose 2B-CL. Membrane-containing fractions were layered onto a linear gradient and spun for 4 h at 27,000 rev m i n - 1 with a SW27 rotor (135,000 g). The fractions were chromatographed on Sephadex G 50 columns and assayed in duplicates in 30 mM MES/Na +, (pH 5.5) 3 mM Na2 ATP, 1.5 mM MgC12 at 18~ C for ATPase activity in the presence of various hormone additions. Wherever the experimental error exceeded the size of the symbols it is indicated by a vertical bar. The Sephadex G 50chromatographed fractions were also tested for various activities. a - # . - ATPase control without additions; Lx-10 8 M I A A ; - : ~ - 1 0 -6 M I A A ; e - 1 0 - 4 M IAA; b o - ATPase control without additions; z~ 10 s M I A A + 1 0 - r [] 10 -6 M I A A + 1 0 - 4 M P A A ; e-10 4MIAA+10 -4MPAA; - i - 10 -4 M PAA; c - 9 cytochrome c oxidase; - o - cytochrome c reductase; A- c~-mannosidase ; - o carotinoids; protein ; d 9 glucan synthetase I; [] glucan synthetase II; o IDPase; -c.,- ATPase without additions

Table 1. Average stimulation of ATPase activity in fractions from a linear sucrose gradient after 4 h centrifugation. Hormone addition

10 -6 M 2,4 D + 1 0 -4 M PAA 10 -6 M 2,3 D + 1 0 -4 M PAA 10 -6 M 3,5 D + 1 0 -4 M PAA

Density range 1.0-1.2 M sucrose

0.6-0.9 M sucrose

54% 33% 31%

41% 3% 6%

on plasma membranes since only they have an acid ATPase, but mitochondria do not (Hodges and Leonard 1972). The activity of ~-mannosidase, a possible marker for vacuoles (Boller and Kende 1979), was low in these dense fractions. In contrast to the rather variable ATPase and glucan synthetase II profiles, the profiles of all other marker enzymes were consistently reproducible. A typical example of the distribution of these enzymes is shown in Fig. 2. Thus, these data may be taken as evidence that at least a portion of the auxin-stimulated ATPase activity was plasma membrane-bound. Part of the difficulties in localizing the auxin-stimulated ATPase may have arisen because after 4-6 h centrifugation all of the membrane particles had not yet reached their equilibrium densities. This conclu-

G.F.E. Scherer: Auxin-stimulated ATPase

437

Fb

a

-15