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Triacontanol stimulates NADH oxidase of soybean hypocotyl plasma membrane
Plant Science, 79 ( 1991 ) 31-36
3I
Elsevier Scientific Publishers Ireland Ltd.
Triacontanol stimulates NADH oxidase of soybean hypocotyl plasma membrane D. J a m e s M o r r 6 a, G u n S e l l d 6 n b, X i a o Z h a n g Z h u a a n d A n d r e w B r i g h t m a n a aDepartment of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907 (U.S.A.) and hBotanical Institute, University of G6teborg, G6teborg (Sweden) (Received April 23rd, 1991; revision received July 2nd, 1991; accepted July 3rd, 1991}
NADH oxidase activity of plasma membranes purified from etiolated hypocotyls of soybean (Glycine max Merr. cv. Williams) was stimulated by about 50% by triacontanol over the concentration range of 10 - 7 - 1 0 -5 M. Small but statistically significant stimulations of elongation of hypocotyl segments also were observed. Octacosanol, a triacontanol antagonist, was without effect on control growth or NADH oxidase activity but inhibited 2,4-dichlorophenoxyacetic acid(2.4-D)-induced growth. We suggest that growth promotions by triacontanol may be mediated by a direct action of triacontanol on the NADH oxidase of the plasma membrane, an enzyme the activity of which previously has been suggested to be rate-limiting to growth.
Key words: auxin; NADH oxidase; plant growth; triacontanol
Introduction
Triacontanol [CHa(CH2)28CH2OH] is a waterinsoluble straight-chain primary alcohol often described as having growth-regulating properties [1]. Because of its solubility properties, a membrane site of action might be anticipated [1]. N A D H oxidase activity of the plasma membrane has been suggested to represent a plant enzyme rate limiting to growth [2]. The activity is stimulated by auxin over the growth promoting range of auxin concentration [3-5]. It is not a peroxidase based on the stoichiometry of oxygen consumed to N A D H oxidized and from the response pattern to inhibitors [6]. NADH oxidase has recently been shown to be activated by fatty acids and lysolipids, products of phospholipase A 2 action [2,7]. These findings prompted us to examine other growth promoCorrespondence to." Dr. D. James Morr6, Hansen Life Sciences Research Building, Department of Medicinal Chemistry & Pharmacognosy, Purdue University, West Lafayette, IN 47907, U.S.A.
ting lipid-soluble substances for effect on the oxidase. Triacontanol was of special interest because of an extensive literature relating to potential for stimulation of plant growth [1]. Materials and Methods
Soybean (Glycine m a x Merr. cv. Williams) seeds were planted in moist vermiculite and grown for 4-5 days in darkness at 24°C. Segments, 1 cm in length, were harvested from the zone of cell elongation under dim laboratory light by cutting 5 mm below the cotyledons. For growth studies, the segments were floated on aqueous solutions, 2 ml/10 sections, containing the test substances. Incubations were in darkness at 24°C for 12-15 h. Lengths were measured to the nearest 0.1 mm. Solvent and detergent mixtures used as vehicles were tested separately. Triacontanol (Polysciences) and octacosanol (Sigma) solutions were emulsions freshly prepared by sonication at a final concentration of 2 mM in 0.01% (w/v) Tween 20 as described [8]. Alternately
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
32
the triacontanol was dissolved in hot D M S O and added directly to the test solutions at a dilution of 1 : 100 or greater. For isolation of plasma membrane, hypocotyl segments were homogenized using a chilled mortar in two volumes (v/w) of a solution containing 25 m M Tris-HC1, 300 m M sucrose, 10 m M KC1 and 1 m M MgC12 (pH 7.5). The homogenates were filtered through a single layer of miracloth to remove debris and cell walls and centrifuged at 8000 x g for 10 min, and the pellets were discarded. The supernatant fractions were then centrifuged at 40 000 x g for 30 min to yield crude microsomal pellets from which plasma membranes were isolated. To isolate plasma membranes, aqueous two-phase partitioning [9] as described by Kjellbom and Larsson [10] was used. Each 16 g system of 6.4% (w/w) polymers (Dextran T500, Pharmacia and Polyethylene Glycol 3350, Fisher)
Triacontanol + T~*en
ab
300
e~ p
Results Tween
0
2oo
X
o -r
I00'
Z
r~
was prepared in a solution containing 0.25 M sucrose and 5 mM potassium phosphate (pH 6.8). The preparations of plasma membranes were monitored by electron microscopy and assay of marker enzymes [11] and were shown to be at least 90% plasma membrane-derived. Freshly isolated plasma membrane vesicles were assayed for N A D H oxidase reaction rates using a spectrophotometric assay to monitor oxidation of N A D H . The reaction mixture contained in a final volume of 2.5 ml, 25 mM T r i s - H C I , 1 mM K C N and 0.1 M sucrose (pH 7.0). The reaction was initiated by the addition of 150 ~zM N A D H . Absorbance was monitored at 340 nm with reference at 430 nm (e : 6.22 x 103 M -I cm -j) using SLM DW 2000 spectrophotometer in the dual wavelength mode of operation. Control preparations contained only membranes and assay buffer plus N A D H and solvents or detergents used as vehicles for the triacontanol. Statistical analyses used the Student's t-test or one way analyses of variance (ANOVA). Tests of least significant difference (LSD) were performed when the F-values were significant (P < 0.05) [12].
0
0
1
I
I
I
I0
2o
30
4o
TIME, MINUTES Fig. 1. Response with time of relative rates of N A D H oxidase activity of plasma membranes from etiolated hypocotyls of soybean to 2 x 10 -6 M triacontanol prepared in 0.01% (10 -4 M) Tween 20 compared to Tween 20 alone. Superimposed upon a time-dependent activation of the activity by the Tween is an initial stimulation by triacontanol. Results are from 3 different membrane preparations 4- S.D. Values followed by different letters are significantly (P < 0.05) different.
N A D H oxidase activity of isolated plasma membrane vesicles was stimulated in a timedependent fashion by Tween 20 at concentrations sufficient to stabilize triacontanol in solution as an emulsion (Fig. 1). Greatest stimulation of the rate of N A D H oxidase occurred between 10 and 20 min of incubation in the presence of detergent alone. Tween 20 to which a near optimal concentration of 2 x 10 -6 M triacontanol had been added elicited a parallel response but with an additional stimulation not given by the detergent alone during the initial 10 min of incubation (Figs. 1 and 2). When examined over a range of triacontanol concentrations, near optimal stimulation was observed between 10 -6 and 10 -5 M (Fig. 2). Elongation growth of 1 cm segments ofetiolated hypocotyls of soybean was also stimulated slightly b y 10 -6 M triacontanol (20% compared to the detergent carrier alone at the same amount as present with the triacontanol) (Fig. 3). Results with D M S O as carrier were similar to
33
0.8
60 acontanol 0.6
I
_ S-1+Io-SM
40 -Triocontenol
8O~
O~
Z
8 20
ab
tr (.9
h 0
ntanol I No Tween
I ] NO
0.2
a_ X 0
0
I
O.C
I
I
-7
I
-6 -5 LOG [TRIACONTANOL], M
Z
I
-4
-20
-40 I
I
-6
-5
LOG [TRIACONTANOL],
M
Dose-response of the triacontanol stimulation of the rate of N A D H oxidase activity from which the response of an appropriate detergent control has been subtracted. Values are from 3 determinations from each of three different membrane preparations ± S.D. among membrane preparations. Values not followed by the same letter were significantly different (P < 0.05) using ANOVA. The variation was due primarily to a variation in the response of the different membrane preparations to detergent of ± 25%.
Fig. 2.
Fig. 3. Growth response ot" 1 cm segments of soybean hypocotyls during incubation for 15 h with and without 10 -5 M 2A-D in the presence or absence of varying concentrations of triacontanol diluted from a 2 mM stock solution prepared in 0.01% Tween 20 (solid curves). The dashed curves show results of identical dilutions of 0.01% Tween 20 but without triacontanol. Results are from 4 determinations with 10 sections measured per determination for each treatment condition ± S.D. among determinations. Values followed by different letters are significantly (P < 0.05) different.
0.6
J~+Triacon1~nol E
o0.4 :E
f .......
u ¢Y (9
those with Tween 20 as carrier (Fig. 4). Growth stimulations by triacontanol in D M S O compared to D M S O alone were smaller than with Tween 20 as carrier but were reproducible especially in the presence of auxin. Concentrations of D M S O of 1% or greater were growth inhibitory. N A D H oxidase activity of the isolated plasma membrane vesicles also was stimulated by triacontanol added in D M S O solution and D M S O alone was without effect (Table I). As with growth (Figs. 3 and 4), the triacontanol stimulation of the oxidase occurred both in the presence and absence of auxin (Table I).
0.2
r
NO D M S O"[
I
-7
JZ,4-D
j,'~.,..~Triecontonol'~
: .ocont=, -
l 0.0
[ .....-~.
7
S" ....
I
""? I
-6 -5 LOG FTRIACONTANOL], M
I
-4
Fig. 4. As in Fig. 3 except that the 2 mM stock solution was prepared by gentle heating at 80°C in pure DMSO prior to dilution in the assay and growth was measured over 12 h. The dashed curves are for dilutions of a DMSO solution prepared in parallel to those containing triacontanol (solid curves). Results are averages of 3 determinations ± S.D. among determinations except for 5 x 10 -7 M and 5 x 10 -6 M which were single determinations.
34 ]'able I. Response to triacontanol and octacosanol of NADH oxidase of isolated vesicles of plasma membrane from soybean hypocotyls in the absence and presence of 2,4-D. Solutions werc prepared in DMSO and added at a final DMSO concentration of 0.1%. This concentration of DMSO was without effect on the NADH oxidase. Controls contained DMSO alone. Results were duplicated using three different membrane preparations. Representative results from a single membrane preparation in which both triacontanol and octacosanol werc compared arc given based on activity averaged over 10 min. n.d., not determined Addition
Conc. (M)
Complete
-
10 -5 M 2,4-D
NADH oxidase nmol(min) -1 (mg prot.) -I
-
0.71 (100%)
+ + +
1.57 (222%) 1.41 (200%) 2.42 (340%) 1.30 (18Y/,,) 2.36 (332%)
10-5
+
10 -6
-
10 -7
+ +
0.89 n.d. 0.79 1.63 0.79 1.63
2 x 10-5
Triacontanol
2 x 10-6 Octacosanol
Octacosanol stimulated the oxidase in the absence of 2,4-D but not in its presence (Table I) and was without effect on growth in the absence o f auxin (Fig. 5). However, in the presence o f auxin, octacosanol exhibited an optimum curve with stimulation at 10 -5 M and strong inhibition at
0.6
a No Tween b
E o
b
....... ./~...... L_ ~~
.~
. ~ . . ( ~c~a~osonol
2,4-D
0.4
-II--
~,.~+
Octacosanol
-Octacosanol 0.2
o.o
"'---~-"
+Oetacosanol _1
_{
_;, LOG rOCTACOSANOL],M
Fig. 5. As in Fig. 3 except using octacosanol. Results are averages of 4 determinations ± S.D. among determinations. Values followed by different letters are significantly (P < 0.05) different.
(125%) (111%) (230%) (I 11%) (230%)
10 -6 M. Neither octacosanol nor triacontanol, however, appeared to interfere with the auxin stimulation of the oxidase. Discussion Triacontanol was first described as a growth regulatory molecule by Ries et al. [13]. Work with the molecule has been somewhat restricted by its extremely low water solubility necessitating primarily use of stable emulsions prepared in the presence of detergent. Our findings show a response o f N A D H oxidase to triacontanol over the same range of concentrations as reported previously to be growth promoting [1]. It is difficult to compare the concentrations employed in solutions where sections are floated to amounts sprayed on intact plants where responses to femtomole doses were reported. Colloidal triacontanol produced a response in corn plants using a spray solution 1 ng/dm 3 triacontanol of solution which was approximately equal to 2.2 x 10 -12 M well below the practical limit for detection of effects on N A D H oxidase. N A D H oxidase is of interest as a component of
35
the redox system of the plasma membrane of both plant and animal cells [2] and, in plants, as an enzymatic activity of the plasma membrane subject to direct stimulation by auxin [3-5]. Because of its low specific activity relative to other redox constituents of the plasma membrane and its sensitivity to auxin hormone regulation, the activity has been suggested to function as a rate limiting enzyme in growth [2]. The stimulation of the oxidase by triacontanol is complicated by a parallel stimulation of the oxidase by the detergent used to stabilize the triacontanol emulsion. The latter increases with time to a maximum after 10-20 min of incubation. A similar increase in activity with time occurs for the activation of the oxidase by fatty acids and lysophospholipids [7] and has been suggested to result from the stabilization of the enzyme in an active conformation. Similar modulations of ATPases of plant and animal membrane systems by lysophosphatidylcholine (lysoPC)+ free fatty acids, or phospholipase A2 have been reviewed by Palmgren et al. [14] and includes work from several groups [15-22]. The H+/K+-ATPase of gastric mucosa is stimulated by lysoPC [22] as are the rates of Ca2+-ATPases [16,18,23]. The products produced by phospholipase A 2 may stabilize the CaZ+-ATPase in a more reactive conformation [23]. Lysophospholipids have been shown to regulate protein kinase C activity from pig brain [24]. Kuroda et al. [25] found that lysophosphatidylinositol 4-monophosphate (LPIP) rose in response to insulin in rat fat cells but attributed this rise in LPIP to a breakdown of phosphatidylinositol 4-phosphate (PIP) via phospholipase A 2. The triacontanol response, in contrast, like that of auxin, appears to occur more rapidly and may involve a more direct effect than that resulting from detergent activation. Triacontanol has been reported to alter activities of several enzymes of sprayed corn seedlings together with dry weight and total protein [26] and to increase growth and photosynthesis CO2 assimilation [27] as well as the total activity of ribulose-bisphosphate carboxylase [28] in Chlamydomonas. Interestingly, a Ca2+-stimulated ATPase of plasma membrane vesicles of barley roots also was stimulated by triacontanol when
treated in the presence of calmodulin [29]. Thus growth effects of triacontanol might be based on activation of plasma membrane-associated activities potentially important to the growth process. Whether or not this activation occurs in a manner distinct from that attributed to products of phospholipase A2 [2,7] has not been determined but does provide a potential mechanism of triacontanol action worthy of further investigation. References l 2
3
4
5
6
7
8
9
l0
ll
S.K. Ries, Regulation of plant growth with triacontanol. CRC Crit. Rev. Plant Sci., 2 (1986) 239-285. D.J. Morr6, A lysolipid cascade in signal transduction cycle of growth control, in: D.J. Mort6, W.F. Boss and F.A. Loewus (Eds.), lnositol Metabolism in Plants, Wiley-Liss, New York, (1990) 227-257. D.J. Mort6, P. Navas, C. Penel and F.J. Castillo, Auxinstimulated NADH oxidase (semidehydroascorbate reductase) of soybean plasma membrane: Role in acidification of cytoplasm? Protoplasma, 133 (1986) 195-197. A.O. Brightman, R. Barr, F.L. Crane and D.J. Mort6, Auxin-stimulated NADH oxidase purified from plasma membrane of soybean. Plant Physiol., 84 (1988) 1264-1269. D.J. Mort6, A.O. Brightman, L+-Y. Wu, R. Barr, B. Leak and F.L. Crane, Role of plasma membrane redox activities in elongation growth in plants. Physiol. Plant, 73 (1988) 187-193. F.L. Crane, R. Barr, A.O. Brightman and D.J. MorrO, The hormone-stimulated NADH oxidase of isolated soybean plasma membrane is not a peroxidase. Plant Physiol., 89 (Suppl.) (1989) l l5. A.O. Brightman, X.-Z. Zhu and D.J. MorrO, Activation of plasma membrane NADH oxidase activity by products of phospholipase A. Plant Physiol., 96 ( 1991 } 1314-1320. R.G. Laughlin, R.L. Munyon, S.K. Ries and V.F. Wert, Growth enhancement of plants by femtomole doses of colloidally dispersed triacontanol. Science. 219 (1983) 1219-1221. P.-A. Albertsson, B. Andersson, C. Larsson and H.-E. Akerlund, Phase partition a method for purification and analysis of cell organelles and membrane vesicles. Methods Biochem. Anal., 28 (1982) 115-150. P. Kjellbom and C. Larsson, Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant, 62 (1984) 501-509. A.S. Sandelius and D.J. Morr6, Plasma membrane isolation methods, in: C. Larsson and I.M+ M@ller (Eds.), The Plant Plasma Membrane - - Structure, Function and Molecular Biology, Springer Verlag, New York, 1990, pp. 44-75.
36 R.R. Sokal and F.J. Rohlf, Biometry: The Principles and Practice of Statistics in Biological Research, 2nd edn, W.H. Freeman and Company, New York, 1981. 13 S.K. Ries, V.F. Wert, C.C. Sweeley and R.A. Leavitt, Triacontanol: a new naturally occurring plant growth regulator. Science, 195 (1977) 1339-1340. 14 M.G. Palmgren, M. Sommarin, P. Ulvskov and P.L. Jorgensen. Modulation of plasma membrane H +-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A 2. Physiol Plant, 74 (1988) 11-19. 15 G. Swoboda, J. Fritzsche and W. Hasselbach, Effects of phospholipase A 2 and albumin on the calcium-dependent ATPase and the lipid composition of sarcoplasmic membranes. Eur. J. Biochem., 95 (1979) 77-88. 16 R.D. Taverna and D.J. Hanahan, Modulation of human erythrocyte Ca-+/Mg-+-ATPase activity by phospholipase A 2 and proteases: A comparison with calmodulin. Biochem. Biophys. Res. Commun., 94 (1980) 652-659. 17 V. Niggli, E.S. Adunyah and E. Carafoli, Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect ofcalmodulin on the purified erythrocyte CaZ+-ATPase. J. Biol. Chem., 256 (1981) 8588-8592. 18 B. Sarkadi, A. Enyedi, A. Nyers and G. Gardos, The function and regulation of the calcium pump in the erythrocyte membrane. Ann. NY. Acad. Sci., 402 (1982) 329-348. 19 G. Schmalzing and P. Kutschera, Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A 2. J. Membrane Biol., 69 (1982) 65-76. 20 A.C. Swann, Free fatty acids and (Na +, K+)-ATPase: Effect of cation regulation, enzyme conformation, and interactions with ethanol. Arch. Biochem. Biophys., 233 (1984) 354-361. 12
21
22
23
24
25
26
27
28
29
R.A. Kelly, C.S. O'Hara, W.E. Mitch and T.W. Smith, Identification of Na+K+-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids. J. Biol. Chem., 261 (1986) 11704-11711. W.B. Im, D.P. Blakeman and J.P. Davis, Effect of lysophosphatidylcholine on K+-transport in rat heavy gastric membranes enriched with (H+-K+-ATPase). Biochem. Biophys. Res. Commun., 146 (1987) 840-848. O. Scharff, B. Foder and U. Skibsted, Hysteretic activation of the Ca 2+ pump revealed by calcium transients in human red cells. Biochim. Biophys. Acta., 730 (1983) 295-305. K. Oishi, R . L Raynor, P.A. Charp and J.F. Kuo, Regulation of protein kinase C by lysophospholipids. J. Biol. Chem., 263 (1983) 6865-6871. Y. Kuroda, H. Nakayama, T. Ishibashi, S. Aoki, S. Tushima and S. Nakagawa, A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells. Fed. Eur. Biochem. Soc.. 224 (1987) 137-141. A.P. Lesniak and S.K. Ries, Changes in enzyme activity of corn seedlings after foliar application of triacontanol. J. Plant Growth Regul., 79 (1983) 121-128. R.L. Houtz, S.K. Ries and N.E. Tolbert, Effect of triacontanol on Chlamydomonas. Plant Physiol., 79 (1985) 357-364. R.L. Houtz, S.K. Ries and N.E. Tolbert, Effect of triacontanol on Chlurnydomonas. II. Specific activity of ribulose-bisphosphate carboxylase/oxygenase, ribulosebisphosphate concentration, and characteristics of photorespiration. Plant Physiol., 79 (1985) 365-370. A.P. Lesniak, A. Haung and S.K. Ries, Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin. Physiol. Plant, 75 (1989) 75-80.
3I
Elsevier Scientific Publishers Ireland Ltd.
Triacontanol stimulates NADH oxidase of soybean hypocotyl plasma membrane D. J a m e s M o r r 6 a, G u n S e l l d 6 n b, X i a o Z h a n g Z h u a a n d A n d r e w B r i g h t m a n a aDepartment of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907 (U.S.A.) and hBotanical Institute, University of G6teborg, G6teborg (Sweden) (Received April 23rd, 1991; revision received July 2nd, 1991; accepted July 3rd, 1991}
NADH oxidase activity of plasma membranes purified from etiolated hypocotyls of soybean (Glycine max Merr. cv. Williams) was stimulated by about 50% by triacontanol over the concentration range of 10 - 7 - 1 0 -5 M. Small but statistically significant stimulations of elongation of hypocotyl segments also were observed. Octacosanol, a triacontanol antagonist, was without effect on control growth or NADH oxidase activity but inhibited 2,4-dichlorophenoxyacetic acid(2.4-D)-induced growth. We suggest that growth promotions by triacontanol may be mediated by a direct action of triacontanol on the NADH oxidase of the plasma membrane, an enzyme the activity of which previously has been suggested to be rate-limiting to growth.
Key words: auxin; NADH oxidase; plant growth; triacontanol
Introduction
Triacontanol [CHa(CH2)28CH2OH] is a waterinsoluble straight-chain primary alcohol often described as having growth-regulating properties [1]. Because of its solubility properties, a membrane site of action might be anticipated [1]. N A D H oxidase activity of the plasma membrane has been suggested to represent a plant enzyme rate limiting to growth [2]. The activity is stimulated by auxin over the growth promoting range of auxin concentration [3-5]. It is not a peroxidase based on the stoichiometry of oxygen consumed to N A D H oxidized and from the response pattern to inhibitors [6]. NADH oxidase has recently been shown to be activated by fatty acids and lysolipids, products of phospholipase A 2 action [2,7]. These findings prompted us to examine other growth promoCorrespondence to." Dr. D. James Morr6, Hansen Life Sciences Research Building, Department of Medicinal Chemistry & Pharmacognosy, Purdue University, West Lafayette, IN 47907, U.S.A.
ting lipid-soluble substances for effect on the oxidase. Triacontanol was of special interest because of an extensive literature relating to potential for stimulation of plant growth [1]. Materials and Methods
Soybean (Glycine m a x Merr. cv. Williams) seeds were planted in moist vermiculite and grown for 4-5 days in darkness at 24°C. Segments, 1 cm in length, were harvested from the zone of cell elongation under dim laboratory light by cutting 5 mm below the cotyledons. For growth studies, the segments were floated on aqueous solutions, 2 ml/10 sections, containing the test substances. Incubations were in darkness at 24°C for 12-15 h. Lengths were measured to the nearest 0.1 mm. Solvent and detergent mixtures used as vehicles were tested separately. Triacontanol (Polysciences) and octacosanol (Sigma) solutions were emulsions freshly prepared by sonication at a final concentration of 2 mM in 0.01% (w/v) Tween 20 as described [8]. Alternately
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
32
the triacontanol was dissolved in hot D M S O and added directly to the test solutions at a dilution of 1 : 100 or greater. For isolation of plasma membrane, hypocotyl segments were homogenized using a chilled mortar in two volumes (v/w) of a solution containing 25 m M Tris-HC1, 300 m M sucrose, 10 m M KC1 and 1 m M MgC12 (pH 7.5). The homogenates were filtered through a single layer of miracloth to remove debris and cell walls and centrifuged at 8000 x g for 10 min, and the pellets were discarded. The supernatant fractions were then centrifuged at 40 000 x g for 30 min to yield crude microsomal pellets from which plasma membranes were isolated. To isolate plasma membranes, aqueous two-phase partitioning [9] as described by Kjellbom and Larsson [10] was used. Each 16 g system of 6.4% (w/w) polymers (Dextran T500, Pharmacia and Polyethylene Glycol 3350, Fisher)
Triacontanol + T~*en
ab
300
e~ p
Results Tween
0
2oo
X
o -r
I00'
Z
r~
was prepared in a solution containing 0.25 M sucrose and 5 mM potassium phosphate (pH 6.8). The preparations of plasma membranes were monitored by electron microscopy and assay of marker enzymes [11] and were shown to be at least 90% plasma membrane-derived. Freshly isolated plasma membrane vesicles were assayed for N A D H oxidase reaction rates using a spectrophotometric assay to monitor oxidation of N A D H . The reaction mixture contained in a final volume of 2.5 ml, 25 mM T r i s - H C I , 1 mM K C N and 0.1 M sucrose (pH 7.0). The reaction was initiated by the addition of 150 ~zM N A D H . Absorbance was monitored at 340 nm with reference at 430 nm (e : 6.22 x 103 M -I cm -j) using SLM DW 2000 spectrophotometer in the dual wavelength mode of operation. Control preparations contained only membranes and assay buffer plus N A D H and solvents or detergents used as vehicles for the triacontanol. Statistical analyses used the Student's t-test or one way analyses of variance (ANOVA). Tests of least significant difference (LSD) were performed when the F-values were significant (P < 0.05) [12].
0
0
1
I
I
I
I0
2o
30
4o
TIME, MINUTES Fig. 1. Response with time of relative rates of N A D H oxidase activity of plasma membranes from etiolated hypocotyls of soybean to 2 x 10 -6 M triacontanol prepared in 0.01% (10 -4 M) Tween 20 compared to Tween 20 alone. Superimposed upon a time-dependent activation of the activity by the Tween is an initial stimulation by triacontanol. Results are from 3 different membrane preparations 4- S.D. Values followed by different letters are significantly (P < 0.05) different.
N A D H oxidase activity of isolated plasma membrane vesicles was stimulated in a timedependent fashion by Tween 20 at concentrations sufficient to stabilize triacontanol in solution as an emulsion (Fig. 1). Greatest stimulation of the rate of N A D H oxidase occurred between 10 and 20 min of incubation in the presence of detergent alone. Tween 20 to which a near optimal concentration of 2 x 10 -6 M triacontanol had been added elicited a parallel response but with an additional stimulation not given by the detergent alone during the initial 10 min of incubation (Figs. 1 and 2). When examined over a range of triacontanol concentrations, near optimal stimulation was observed between 10 -6 and 10 -5 M (Fig. 2). Elongation growth of 1 cm segments ofetiolated hypocotyls of soybean was also stimulated slightly b y 10 -6 M triacontanol (20% compared to the detergent carrier alone at the same amount as present with the triacontanol) (Fig. 3). Results with D M S O as carrier were similar to
33
0.8
60 acontanol 0.6
I
_ S-1+Io-SM
40 -Triocontenol
8O~
O~
Z
8 20
ab
tr (.9
h 0
ntanol I No Tween
I ] NO
0.2
a_ X 0
0
I
O.C
I
I
-7
I
-6 -5 LOG [TRIACONTANOL], M
Z
I
-4
-20
-40 I
I
-6
-5
LOG [TRIACONTANOL],
M
Dose-response of the triacontanol stimulation of the rate of N A D H oxidase activity from which the response of an appropriate detergent control has been subtracted. Values are from 3 determinations from each of three different membrane preparations ± S.D. among membrane preparations. Values not followed by the same letter were significantly different (P < 0.05) using ANOVA. The variation was due primarily to a variation in the response of the different membrane preparations to detergent of ± 25%.
Fig. 2.
Fig. 3. Growth response ot" 1 cm segments of soybean hypocotyls during incubation for 15 h with and without 10 -5 M 2A-D in the presence or absence of varying concentrations of triacontanol diluted from a 2 mM stock solution prepared in 0.01% Tween 20 (solid curves). The dashed curves show results of identical dilutions of 0.01% Tween 20 but without triacontanol. Results are from 4 determinations with 10 sections measured per determination for each treatment condition ± S.D. among determinations. Values followed by different letters are significantly (P < 0.05) different.
0.6
J~+Triacon1~nol E
o0.4 :E
f .......
u ¢Y (9
those with Tween 20 as carrier (Fig. 4). Growth stimulations by triacontanol in D M S O compared to D M S O alone were smaller than with Tween 20 as carrier but were reproducible especially in the presence of auxin. Concentrations of D M S O of 1% or greater were growth inhibitory. N A D H oxidase activity of the isolated plasma membrane vesicles also was stimulated by triacontanol added in D M S O solution and D M S O alone was without effect (Table I). As with growth (Figs. 3 and 4), the triacontanol stimulation of the oxidase occurred both in the presence and absence of auxin (Table I).
0.2
r
NO D M S O"[
I
-7
JZ,4-D
j,'~.,..~Triecontonol'~
: .ocont=, -
l 0.0
[ .....-~.
7
S" ....
I
""? I
-6 -5 LOG FTRIACONTANOL], M
I
-4
Fig. 4. As in Fig. 3 except that the 2 mM stock solution was prepared by gentle heating at 80°C in pure DMSO prior to dilution in the assay and growth was measured over 12 h. The dashed curves are for dilutions of a DMSO solution prepared in parallel to those containing triacontanol (solid curves). Results are averages of 3 determinations ± S.D. among determinations except for 5 x 10 -7 M and 5 x 10 -6 M which were single determinations.
34 ]'able I. Response to triacontanol and octacosanol of NADH oxidase of isolated vesicles of plasma membrane from soybean hypocotyls in the absence and presence of 2,4-D. Solutions werc prepared in DMSO and added at a final DMSO concentration of 0.1%. This concentration of DMSO was without effect on the NADH oxidase. Controls contained DMSO alone. Results were duplicated using three different membrane preparations. Representative results from a single membrane preparation in which both triacontanol and octacosanol werc compared arc given based on activity averaged over 10 min. n.d., not determined Addition
Conc. (M)
Complete
-
10 -5 M 2,4-D
NADH oxidase nmol(min) -1 (mg prot.) -I
-
0.71 (100%)
+ + +
1.57 (222%) 1.41 (200%) 2.42 (340%) 1.30 (18Y/,,) 2.36 (332%)
10-5
+
10 -6
-
10 -7
+ +
0.89 n.d. 0.79 1.63 0.79 1.63
2 x 10-5
Triacontanol
2 x 10-6 Octacosanol
Octacosanol stimulated the oxidase in the absence of 2,4-D but not in its presence (Table I) and was without effect on growth in the absence o f auxin (Fig. 5). However, in the presence o f auxin, octacosanol exhibited an optimum curve with stimulation at 10 -5 M and strong inhibition at
0.6
a No Tween b
E o
b
....... ./~...... L_ ~~
.~
. ~ . . ( ~c~a~osonol
2,4-D
0.4
-II--
~,.~+
Octacosanol
-Octacosanol 0.2
o.o
"'---~-"
+Oetacosanol _1
_{
_;, LOG rOCTACOSANOL],M
Fig. 5. As in Fig. 3 except using octacosanol. Results are averages of 4 determinations ± S.D. among determinations. Values followed by different letters are significantly (P < 0.05) different.
(125%) (111%) (230%) (I 11%) (230%)
10 -6 M. Neither octacosanol nor triacontanol, however, appeared to interfere with the auxin stimulation of the oxidase. Discussion Triacontanol was first described as a growth regulatory molecule by Ries et al. [13]. Work with the molecule has been somewhat restricted by its extremely low water solubility necessitating primarily use of stable emulsions prepared in the presence of detergent. Our findings show a response o f N A D H oxidase to triacontanol over the same range of concentrations as reported previously to be growth promoting [1]. It is difficult to compare the concentrations employed in solutions where sections are floated to amounts sprayed on intact plants where responses to femtomole doses were reported. Colloidal triacontanol produced a response in corn plants using a spray solution 1 ng/dm 3 triacontanol of solution which was approximately equal to 2.2 x 10 -12 M well below the practical limit for detection of effects on N A D H oxidase. N A D H oxidase is of interest as a component of
35
the redox system of the plasma membrane of both plant and animal cells [2] and, in plants, as an enzymatic activity of the plasma membrane subject to direct stimulation by auxin [3-5]. Because of its low specific activity relative to other redox constituents of the plasma membrane and its sensitivity to auxin hormone regulation, the activity has been suggested to function as a rate limiting enzyme in growth [2]. The stimulation of the oxidase by triacontanol is complicated by a parallel stimulation of the oxidase by the detergent used to stabilize the triacontanol emulsion. The latter increases with time to a maximum after 10-20 min of incubation. A similar increase in activity with time occurs for the activation of the oxidase by fatty acids and lysophospholipids [7] and has been suggested to result from the stabilization of the enzyme in an active conformation. Similar modulations of ATPases of plant and animal membrane systems by lysophosphatidylcholine (lysoPC)+ free fatty acids, or phospholipase A2 have been reviewed by Palmgren et al. [14] and includes work from several groups [15-22]. The H+/K+-ATPase of gastric mucosa is stimulated by lysoPC [22] as are the rates of Ca2+-ATPases [16,18,23]. The products produced by phospholipase A 2 may stabilize the CaZ+-ATPase in a more reactive conformation [23]. Lysophospholipids have been shown to regulate protein kinase C activity from pig brain [24]. Kuroda et al. [25] found that lysophosphatidylinositol 4-monophosphate (LPIP) rose in response to insulin in rat fat cells but attributed this rise in LPIP to a breakdown of phosphatidylinositol 4-phosphate (PIP) via phospholipase A 2. The triacontanol response, in contrast, like that of auxin, appears to occur more rapidly and may involve a more direct effect than that resulting from detergent activation. Triacontanol has been reported to alter activities of several enzymes of sprayed corn seedlings together with dry weight and total protein [26] and to increase growth and photosynthesis CO2 assimilation [27] as well as the total activity of ribulose-bisphosphate carboxylase [28] in Chlamydomonas. Interestingly, a Ca2+-stimulated ATPase of plasma membrane vesicles of barley roots also was stimulated by triacontanol when
treated in the presence of calmodulin [29]. Thus growth effects of triacontanol might be based on activation of plasma membrane-associated activities potentially important to the growth process. Whether or not this activation occurs in a manner distinct from that attributed to products of phospholipase A2 [2,7] has not been determined but does provide a potential mechanism of triacontanol action worthy of further investigation. References l 2
3
4
5
6
7
8
9
l0
ll
S.K. Ries, Regulation of plant growth with triacontanol. CRC Crit. Rev. Plant Sci., 2 (1986) 239-285. D.J. Morr6, A lysolipid cascade in signal transduction cycle of growth control, in: D.J. Mort6, W.F. Boss and F.A. Loewus (Eds.), lnositol Metabolism in Plants, Wiley-Liss, New York, (1990) 227-257. D.J. Mort6, P. Navas, C. Penel and F.J. Castillo, Auxinstimulated NADH oxidase (semidehydroascorbate reductase) of soybean plasma membrane: Role in acidification of cytoplasm? Protoplasma, 133 (1986) 195-197. A.O. Brightman, R. Barr, F.L. Crane and D.J. Mort6, Auxin-stimulated NADH oxidase purified from plasma membrane of soybean. Plant Physiol., 84 (1988) 1264-1269. D.J. Mort6, A.O. Brightman, L+-Y. Wu, R. Barr, B. Leak and F.L. Crane, Role of plasma membrane redox activities in elongation growth in plants. Physiol. Plant, 73 (1988) 187-193. F.L. Crane, R. Barr, A.O. Brightman and D.J. MorrO, The hormone-stimulated NADH oxidase of isolated soybean plasma membrane is not a peroxidase. Plant Physiol., 89 (Suppl.) (1989) l l5. A.O. Brightman, X.-Z. Zhu and D.J. MorrO, Activation of plasma membrane NADH oxidase activity by products of phospholipase A. Plant Physiol., 96 ( 1991 } 1314-1320. R.G. Laughlin, R.L. Munyon, S.K. Ries and V.F. Wert, Growth enhancement of plants by femtomole doses of colloidally dispersed triacontanol. Science. 219 (1983) 1219-1221. P.-A. Albertsson, B. Andersson, C. Larsson and H.-E. Akerlund, Phase partition a method for purification and analysis of cell organelles and membrane vesicles. Methods Biochem. Anal., 28 (1982) 115-150. P. Kjellbom and C. Larsson, Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant, 62 (1984) 501-509. A.S. Sandelius and D.J. Morr6, Plasma membrane isolation methods, in: C. Larsson and I.M+ M@ller (Eds.), The Plant Plasma Membrane - - Structure, Function and Molecular Biology, Springer Verlag, New York, 1990, pp. 44-75.
36 R.R. Sokal and F.J. Rohlf, Biometry: The Principles and Practice of Statistics in Biological Research, 2nd edn, W.H. Freeman and Company, New York, 1981. 13 S.K. Ries, V.F. Wert, C.C. Sweeley and R.A. Leavitt, Triacontanol: a new naturally occurring plant growth regulator. Science, 195 (1977) 1339-1340. 14 M.G. Palmgren, M. Sommarin, P. Ulvskov and P.L. Jorgensen. Modulation of plasma membrane H +-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A 2. Physiol Plant, 74 (1988) 11-19. 15 G. Swoboda, J. Fritzsche and W. Hasselbach, Effects of phospholipase A 2 and albumin on the calcium-dependent ATPase and the lipid composition of sarcoplasmic membranes. Eur. J. Biochem., 95 (1979) 77-88. 16 R.D. Taverna and D.J. Hanahan, Modulation of human erythrocyte Ca-+/Mg-+-ATPase activity by phospholipase A 2 and proteases: A comparison with calmodulin. Biochem. Biophys. Res. Commun., 94 (1980) 652-659. 17 V. Niggli, E.S. Adunyah and E. Carafoli, Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect ofcalmodulin on the purified erythrocyte CaZ+-ATPase. J. Biol. Chem., 256 (1981) 8588-8592. 18 B. Sarkadi, A. Enyedi, A. Nyers and G. Gardos, The function and regulation of the calcium pump in the erythrocyte membrane. Ann. NY. Acad. Sci., 402 (1982) 329-348. 19 G. Schmalzing and P. Kutschera, Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A 2. J. Membrane Biol., 69 (1982) 65-76. 20 A.C. Swann, Free fatty acids and (Na +, K+)-ATPase: Effect of cation regulation, enzyme conformation, and interactions with ethanol. Arch. Biochem. Biophys., 233 (1984) 354-361. 12
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R.A. Kelly, C.S. O'Hara, W.E. Mitch and T.W. Smith, Identification of Na+K+-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids. J. Biol. Chem., 261 (1986) 11704-11711. W.B. Im, D.P. Blakeman and J.P. Davis, Effect of lysophosphatidylcholine on K+-transport in rat heavy gastric membranes enriched with (H+-K+-ATPase). Biochem. Biophys. Res. Commun., 146 (1987) 840-848. O. Scharff, B. Foder and U. Skibsted, Hysteretic activation of the Ca 2+ pump revealed by calcium transients in human red cells. Biochim. Biophys. Acta., 730 (1983) 295-305. K. Oishi, R . L Raynor, P.A. Charp and J.F. Kuo, Regulation of protein kinase C by lysophospholipids. J. Biol. Chem., 263 (1983) 6865-6871. Y. Kuroda, H. Nakayama, T. Ishibashi, S. Aoki, S. Tushima and S. Nakagawa, A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells. Fed. Eur. Biochem. Soc.. 224 (1987) 137-141. A.P. Lesniak and S.K. Ries, Changes in enzyme activity of corn seedlings after foliar application of triacontanol. J. Plant Growth Regul., 79 (1983) 121-128. R.L. Houtz, S.K. Ries and N.E. Tolbert, Effect of triacontanol on Chlamydomonas. Plant Physiol., 79 (1985) 357-364. R.L. Houtz, S.K. Ries and N.E. Tolbert, Effect of triacontanol on Chlurnydomonas. II. Specific activity of ribulose-bisphosphate carboxylase/oxygenase, ribulosebisphosphate concentration, and characteristics of photorespiration. Plant Physiol., 79 (1985) 365-370. A.P. Lesniak, A. Haung and S.K. Ries, Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin. Physiol. Plant, 75 (1989) 75-80.