Planta (Berl.) 122, 79--90 (1975) 9 by Springer-Verlag 1975
Inhibition of Ion Accumulation in Maize Roots by Abscisic Acid Dale L. Shaner, Stuart M. Mertz, Jr.*, and Charles J. Arntzen Department of Botany, University of Illinois, Urbana, Illinois 61801, USA Received 12 September; accepted 1 November, 1974 Summary. An inhibition of root growth, a decrease in the amount of potassium (as S6Rb) and phosphate (32p) accumulation by the root, and a partial depolarization of transmembrane electropotential were observed to develop with a similar time course and to a similar extent when intact maize (Zea mays L.) roots were treated with 10-3 M abseisic acid (ABA). Potassium uptake was inhibited by ABA when excised, low-salt roots were bathed in KC1, KH2P04, or K3SO4. ABA did not affect the ATP content of the tissues, the activity of isolated mitochondria, nor the activity of mierosomal K+-stimulated ATPases.
Introduction Abseisie acid (ABA) is known to inhibit plant growth, in some cases with a very rapid response (Warner and Leopold, 1971; Rehm and Cline, 1973; Milborrow, 1974). Attempts have been made to understand its mechanism(s) of action in elieiting this response. Several effects on cellular metabolism have been reported, including a reduction in rates of protein and nucleic-acid synthesis, although these cannot be detected until after 2 or more hours of ABA treatment (Milborrow, 1974). Van Steveninek (1972), Cram and Pitman (1972) and Pitman and Cram (1973) have suggested that ABA may function as a hormonal regulator of ion-transport processes in plant cells. The most rapid and dramatic effect in this respect is the well-characterized induction of stomatal closure observed after ABA application to whole leaves: this is thought to be due to an inhibitory action of ABA on K+ uptake by guard cells (Cummins et al., 1971; Mansfield and Jones, 1971 ; I-Iorton, 1971 ; Arntzen et al., 1973). Reports on the effect of ABA on ion transport into other plant tissues are somewhat inconsistent. Mansfield and Jones (1971) found ABA inhibited K+ uptake by leaves; Cram and Pitman (1972) and Collins and Kerrigan (1973) reported that it stimulated K+ uptake into roots. I n aged beet-root discs, ABA temporarily inhibited K + uptake, stimulated Na + and CI- uptake, and altered the K/Na selectivity (Van Steveninek, 1972). Some of these different results may in part be due to different experimental conditions. We will describe evidence demonstrating that ABA does inhibit K+ uptake into roots, with effects being evident as early as 30 rain after treatment. The inhibition of root growth observed by treating roots with the growth regulator will be correlated with the extent of inhibition of ion uptake and the change in electrochemical potential of cell membranes. * Present address: Monsanto CommercialProducts Co., Agricultural Division, 800 N. Lindbergh Blvd., St. Louis, Missouri 63166, USA.
80
D . L . Shaner et al. Material and Methods
Plant Material. Seeds of Zea mays L. (WF9 • M14) (Crow Hybrids, Melford, Ill., USA) were surface-sterilized in 1% NaOC1 for 15 rain, rinsed, and germinated in enamel pans on paper towels soaked with 0.5 mM CaS0 v The pans were covered with plastic food-wrap which was slit in several places to allow air exchange. After 2 days in darkness a t 28 ~ the seedlings were transferred to large-mesh plastic screens on beakers so t h a t roots extended down into well-aerated 0.5 mM CaSOa for 1 additional day. Primary roots of 3-day-old seedlings were used in all experiments. Ion Uptake. CaSOd-grown roots were specifically chosen for these studies since the behavior of these roots in various salt solutions is well documented in the literature, l~oots so grown contain low amounts of ions. This " l o w - s a l t " condition is i m p o r t a n t because radiotracer uptake occurs into all the various compartments of the cell a n d root. I n addition, we have used short uptake times (30 min or less) to insure t h a t radiotracer uptake is nearly equivalent to the true n e t uptake since the specific activity of the ion in the uptake solution is not greatly diluted b y ions in the cell, a n d efflux is a t a minimum. Calcium was included in all solutions (0.5 mM Ca 2+) because it is known to play an i m p o r t a n t role in selective ion accumulation (see Epstein, 1972). For measurement of ion uptake into roots of intact seedlings, samples of plants growing on screens were simply lifted on the screen, a n d transferred to isotope-containing solutions in 1-1 beakers. After the appropriate uptake period, 30 seedlings were transferred to screens over cold (4 ~) solutions identical to those used for uptake b u t not containing isotope, for a 30-rain desorption of exchangable isotope. The terminal 2.5 cm segments of the primary roots were t h e n quickly excised, blotted dry, weighed, a n d placed in scintillation vials. I n ion-uptake studies with excised roots, terminal 2.5 cm segments of 15 primary roots of CaSOa-grown plants were excised and placed in 100 ml solution in 500 ml flasks. Following t h e uptake period, the roots were transferred to cold (4 ~) isotope-free b u t otherwise identical solutions a n d shaken for a 30 rain desorption period. They were t h e n blotted dry, weighed, and placed in scintillation vials. Each scintillation vial contained 15 ml of a scintillation cocktail (886 ml dioxane, 134 ml methylcellosolve, 50 g naphthalene, 4 g 2,5-diphenyloxazole, 50 mg p-bis-[2-(5-phenoyloxazolyl)]-benzene, and 60 g Cab-O-Sil thixotrophic gel per liter of cocktail (Bruno a n d Christian, 1961). The activity was counted in a Packard TriCarb liquid scintillation counter. Each ion-uptake experiment was done with duplicate or triplicate samples and was repeated 2 or more times. The results presented are representative of consistent patterns of ABA effects. Measurement el A T P Content. The initial procedures for the t r e a t m e n t of plants were identical to those for the studies of ion uptake into intact roots, b u t the solutions were without radiotracers. After the appropriate incubation period, duplicate samples of 20 roots were excised, blotted dry, and weighed. The sections were extracted in 10 ml boiling 0.02 M glycine buffer (pH 7.7), and analyzed for A T P content b y the luciferin-luciferase technique as described b y Lin a n d Hanson (1974). The studies were conducted in Prof. J. B. Hanson's laboratory in this Department and their control procedures were used to demonstrate maximal ATP recovery. Isolation and Assay o] Mitochondria. Mitochondria were isolated from maize seedlings a n d resuspended b y the procedures of Miller et al. (1970). Measurement of the rate of O 2 uptake was made with a Clark oxygen electrode in 4 ml reaction solution (274-0.2 ~ containing 0.3 M sucrose, 0 . 0 2 M Tris-HC[ (pH 7.5), 0.004M KHzPO 4 a n d 1 mg/ml bovine serum albumin. Light transmittance of the mitochondrial mixture was measured b y fitting the reaction vessel in the light p a t h of a modified Bausch and Lomb Spectronic 70 spectrephotometer. 02 and percent transmittance changes were monitored simultaneously on a daMpen recorder. Protein was determined b y the method of Lowry et al. (1951). Assay o] Microsomal A TPases. Microsomal membranes were isolated b y the procedure of Leonard and Hedges (1973) except 2.5 cm terminal tips of 3-day-old roots were used, the grinding solution contained 5 0 r a M Tris-MES buffer, and the sucrose-gradient step was omitted. The ATPase-assay reaction-mixture contained 10 mM MES with sufficient Tris buffer added to bring the p H to 6.5, 3 mM MgSOd, 3 mM Tris-ATP and 50 ~zg m e m b r a n e protein in a volume of 1 ml. Oligomycin, which was found to have negligible effect on the K+-stimulated activity in other experiments, was added in all tubes at 5 ~zg/ml to inhibit
Inhibition of I o n Accumulation b y ABA
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Fig. 1 A a n d B. Effects of ABA on elongation of roots on intact seedlings over short (A) a n d long (B) time periods. [] = no ABA; 9 = 10-5 M ABA, o = 10-a M ABA. CaS0a-grown seedlings (see Material and Methods) were transferred on screens to aerated solutions of 1.0 mM KH2PO 4 4- 0.5 mM CaSO~ ~: ABA. Root length was measured from seed to root tip a t t h e intervals indicated. Changes in root length after the time of transfer are shown. E a c h point is t h e average of measurements of 20 roots. Vertical bars a t each point -----s t a n d a r d error of t h e mean (if larger t h a n symbol size)
mitochondrial ATPase. Membranes were incubated with 2 • 1O-~ M A B A (pH 6.5) for 15 rain a t room temperature in a volume of 0.5 ml prior to addition of all other reaction components. The reaction was r u n 30 min a t 37% Measurement o] Transmembrane Electri~ Potential Di/]erence (PD). To obtain measurem e n t s of m e m b r a n e P D under conditions identical to t h e root-growth a n d ion-upteke experiments, determinations of P D were made from cells in t h e region 1 to 2 cm from the tip of a freshly-excised root segment 2.5 cm long. Mertz (1973) found t h a t a t a distance several cell-lengths away from the cut surface, the P D of cells of freshly-excised segments of barley roots was equal to t h a t found in cells of roots of i n t a c t seedlings. The segment was placed in a plexiglass chamber designed to hold it horizontal, a n d was continuously washed with fresh solution (Mertz, 1973). The chamber was m o u n t e d on a microscope stage, thus allowing visual observation of the insertion of t h e microelectrode into epidermal cells. P D was continuously recorded during electrode insertion a n d during long-term measurements, including periods of sample change (e.g., addition of ABA or l~aNa). All measurements were made within 30 rain from time of excision. PDs reported here are from epidermal cells; measurem e n t s are from vacuole to external solution. Electrical measurements were made using the following equipment: 3 M KCl-filled, glass micropipettes (2% agar in reference pipette); Ag/AgC1 electrodes; a n d Sargent-Welch (Chicago, IlL, USA) Model L S X pH/millivolt meter, a n d Model S R G Potentiometric Recorder. Solutions. A stock solution of 10-a M ABA was prepared b y dissolving 25 m g of dry ABA trans-ABA; Sigma Biochem. Co., St. Louis, MO., USA] in i ml of absolute ethanol, [(• a n d t h e n adding 96 ml of deionized distilled water. The p H of these solution was adjusted to 5.6 with 1 I~ KOH. The solution was stored a t 5 ~ in a n aluminium-foil-covered flask for no longer t h a n 5 days. All solutions were adjusted to p H 5.6 a n d a temperature of 24 ~ unless stated otherwise. All root b a t h i n g solutions were aerated. Most experiments utilized a two-salt solution consisting of 0.5 mM CaS04 and KC1, K~SOa, or KH2PO a. The designation KH2PO a actually represents a phosphate-buffer solution prepared b y mixing 0.1 M K2HPO a and 0.1 M KH~PO 4 in the ratio 5:95, a n d diluting to the appropriate PO a concentration (0.1 or 1 raM). 6
Planta
(Berl.), Vol. 122
D.L. Shaner et aL
82
/s
C~176/,,,"~
I
2 Time (hr)
3
4
Fig. 2. Effect of 10-5 M ABA on potassium (SeRb) accumulation from 1 mM KH~PO4+ 0.5 mM CaSO4. o = no ABA, A = 10-~ M ABA. Conditions identical to those described for Fig. 1, except S6Rb+ was included in the root bathing solution. Vertical bars = one standard deviation (if larger than symbol size}
Results 1. E]lects of A B A on Growth and Ion Uptake We have examined the effect of ABA on root growth of maize seedlings over short and long time intervals (Fig. 1A and B, respectively). 10-4 M ABA inhibited root growth b y 56% over a 25-h period {Fig. 1B). The rate of growth at each ABA level was constant from 5 to 25 h. The amount of growth between treated and control roots was significantly different (22%; P = 0.05) at both 10 -4 and 10-5 M ABA after 2 h of treatment. We have attempted to determine whether changes in salt uptake by the ABA-treated roots occurred simultaneously with or prior to the observed time of root-growth inhibition. This was studied b y monitoring the uptake of either SeRb-labeled potassium or a'P-labeled phosphate from a 1.0 mM KH2PO , solution identical to that used in the r o o t - g r o ~ h experiment. Intact seedlings were used during the period of isotope uptake. A significant inhibition (P < 0.05) of both potassium (19%) and phosphate (13%) uptake was observed within 2 h in the presence of 10-5 M ABA (Figs. 2, 3). Experiments to characterize the inhibitory effect of ABA on potassium and phosphate uptake more fully were conducted with excised low-salt roots. I n Table 1 it can be seen tha~ K + accumulation from a KC1 solution is also inhibited b y ABA. A significant inhibition (P
Inhibition of Ion Accumulation in Maize Roots by Abscisic Acid Dale L. Shaner, Stuart M. Mertz, Jr.*, and Charles J. Arntzen Department of Botany, University of Illinois, Urbana, Illinois 61801, USA Received 12 September; accepted 1 November, 1974 Summary. An inhibition of root growth, a decrease in the amount of potassium (as S6Rb) and phosphate (32p) accumulation by the root, and a partial depolarization of transmembrane electropotential were observed to develop with a similar time course and to a similar extent when intact maize (Zea mays L.) roots were treated with 10-3 M abseisic acid (ABA). Potassium uptake was inhibited by ABA when excised, low-salt roots were bathed in KC1, KH2P04, or K3SO4. ABA did not affect the ATP content of the tissues, the activity of isolated mitochondria, nor the activity of mierosomal K+-stimulated ATPases.
Introduction Abseisie acid (ABA) is known to inhibit plant growth, in some cases with a very rapid response (Warner and Leopold, 1971; Rehm and Cline, 1973; Milborrow, 1974). Attempts have been made to understand its mechanism(s) of action in elieiting this response. Several effects on cellular metabolism have been reported, including a reduction in rates of protein and nucleic-acid synthesis, although these cannot be detected until after 2 or more hours of ABA treatment (Milborrow, 1974). Van Steveninek (1972), Cram and Pitman (1972) and Pitman and Cram (1973) have suggested that ABA may function as a hormonal regulator of ion-transport processes in plant cells. The most rapid and dramatic effect in this respect is the well-characterized induction of stomatal closure observed after ABA application to whole leaves: this is thought to be due to an inhibitory action of ABA on K+ uptake by guard cells (Cummins et al., 1971; Mansfield and Jones, 1971 ; I-Iorton, 1971 ; Arntzen et al., 1973). Reports on the effect of ABA on ion transport into other plant tissues are somewhat inconsistent. Mansfield and Jones (1971) found ABA inhibited K+ uptake by leaves; Cram and Pitman (1972) and Collins and Kerrigan (1973) reported that it stimulated K+ uptake into roots. I n aged beet-root discs, ABA temporarily inhibited K + uptake, stimulated Na + and CI- uptake, and altered the K/Na selectivity (Van Steveninek, 1972). Some of these different results may in part be due to different experimental conditions. We will describe evidence demonstrating that ABA does inhibit K+ uptake into roots, with effects being evident as early as 30 rain after treatment. The inhibition of root growth observed by treating roots with the growth regulator will be correlated with the extent of inhibition of ion uptake and the change in electrochemical potential of cell membranes. * Present address: Monsanto CommercialProducts Co., Agricultural Division, 800 N. Lindbergh Blvd., St. Louis, Missouri 63166, USA.
80
D . L . Shaner et al. Material and Methods
Plant Material. Seeds of Zea mays L. (WF9 • M14) (Crow Hybrids, Melford, Ill., USA) were surface-sterilized in 1% NaOC1 for 15 rain, rinsed, and germinated in enamel pans on paper towels soaked with 0.5 mM CaS0 v The pans were covered with plastic food-wrap which was slit in several places to allow air exchange. After 2 days in darkness a t 28 ~ the seedlings were transferred to large-mesh plastic screens on beakers so t h a t roots extended down into well-aerated 0.5 mM CaSOa for 1 additional day. Primary roots of 3-day-old seedlings were used in all experiments. Ion Uptake. CaSOd-grown roots were specifically chosen for these studies since the behavior of these roots in various salt solutions is well documented in the literature, l~oots so grown contain low amounts of ions. This " l o w - s a l t " condition is i m p o r t a n t because radiotracer uptake occurs into all the various compartments of the cell a n d root. I n addition, we have used short uptake times (30 min or less) to insure t h a t radiotracer uptake is nearly equivalent to the true n e t uptake since the specific activity of the ion in the uptake solution is not greatly diluted b y ions in the cell, a n d efflux is a t a minimum. Calcium was included in all solutions (0.5 mM Ca 2+) because it is known to play an i m p o r t a n t role in selective ion accumulation (see Epstein, 1972). For measurement of ion uptake into roots of intact seedlings, samples of plants growing on screens were simply lifted on the screen, a n d transferred to isotope-containing solutions in 1-1 beakers. After the appropriate uptake period, 30 seedlings were transferred to screens over cold (4 ~) solutions identical to those used for uptake b u t not containing isotope, for a 30-rain desorption of exchangable isotope. The terminal 2.5 cm segments of the primary roots were t h e n quickly excised, blotted dry, weighed, a n d placed in scintillation vials. I n ion-uptake studies with excised roots, terminal 2.5 cm segments of 15 primary roots of CaSOa-grown plants were excised and placed in 100 ml solution in 500 ml flasks. Following t h e uptake period, the roots were transferred to cold (4 ~) isotope-free b u t otherwise identical solutions a n d shaken for a 30 rain desorption period. They were t h e n blotted dry, weighed, and placed in scintillation vials. Each scintillation vial contained 15 ml of a scintillation cocktail (886 ml dioxane, 134 ml methylcellosolve, 50 g naphthalene, 4 g 2,5-diphenyloxazole, 50 mg p-bis-[2-(5-phenoyloxazolyl)]-benzene, and 60 g Cab-O-Sil thixotrophic gel per liter of cocktail (Bruno a n d Christian, 1961). The activity was counted in a Packard TriCarb liquid scintillation counter. Each ion-uptake experiment was done with duplicate or triplicate samples and was repeated 2 or more times. The results presented are representative of consistent patterns of ABA effects. Measurement el A T P Content. The initial procedures for the t r e a t m e n t of plants were identical to those for the studies of ion uptake into intact roots, b u t the solutions were without radiotracers. After the appropriate incubation period, duplicate samples of 20 roots were excised, blotted dry, and weighed. The sections were extracted in 10 ml boiling 0.02 M glycine buffer (pH 7.7), and analyzed for A T P content b y the luciferin-luciferase technique as described b y Lin a n d Hanson (1974). The studies were conducted in Prof. J. B. Hanson's laboratory in this Department and their control procedures were used to demonstrate maximal ATP recovery. Isolation and Assay o] Mitochondria. Mitochondria were isolated from maize seedlings a n d resuspended b y the procedures of Miller et al. (1970). Measurement of the rate of O 2 uptake was made with a Clark oxygen electrode in 4 ml reaction solution (274-0.2 ~ containing 0.3 M sucrose, 0 . 0 2 M Tris-HC[ (pH 7.5), 0.004M KHzPO 4 a n d 1 mg/ml bovine serum albumin. Light transmittance of the mitochondrial mixture was measured b y fitting the reaction vessel in the light p a t h of a modified Bausch and Lomb Spectronic 70 spectrephotometer. 02 and percent transmittance changes were monitored simultaneously on a daMpen recorder. Protein was determined b y the method of Lowry et al. (1951). Assay o] Microsomal A TPases. Microsomal membranes were isolated b y the procedure of Leonard and Hedges (1973) except 2.5 cm terminal tips of 3-day-old roots were used, the grinding solution contained 5 0 r a M Tris-MES buffer, and the sucrose-gradient step was omitted. The ATPase-assay reaction-mixture contained 10 mM MES with sufficient Tris buffer added to bring the p H to 6.5, 3 mM MgSOd, 3 mM Tris-ATP and 50 ~zg m e m b r a n e protein in a volume of 1 ml. Oligomycin, which was found to have negligible effect on the K+-stimulated activity in other experiments, was added in all tubes at 5 ~zg/ml to inhibit
Inhibition of I o n Accumulation b y ABA
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4 Time (hr}
6
Fig. 1 A a n d B. Effects of ABA on elongation of roots on intact seedlings over short (A) a n d long (B) time periods. [] = no ABA; 9 = 10-5 M ABA, o = 10-a M ABA. CaS0a-grown seedlings (see Material and Methods) were transferred on screens to aerated solutions of 1.0 mM KH2PO 4 4- 0.5 mM CaSO~ ~: ABA. Root length was measured from seed to root tip a t t h e intervals indicated. Changes in root length after the time of transfer are shown. E a c h point is t h e average of measurements of 20 roots. Vertical bars a t each point -----s t a n d a r d error of t h e mean (if larger t h a n symbol size)
mitochondrial ATPase. Membranes were incubated with 2 • 1O-~ M A B A (pH 6.5) for 15 rain a t room temperature in a volume of 0.5 ml prior to addition of all other reaction components. The reaction was r u n 30 min a t 37% Measurement o] Transmembrane Electri~ Potential Di/]erence (PD). To obtain measurem e n t s of m e m b r a n e P D under conditions identical to t h e root-growth a n d ion-upteke experiments, determinations of P D were made from cells in t h e region 1 to 2 cm from the tip of a freshly-excised root segment 2.5 cm long. Mertz (1973) found t h a t a t a distance several cell-lengths away from the cut surface, the P D of cells of freshly-excised segments of barley roots was equal to t h a t found in cells of roots of i n t a c t seedlings. The segment was placed in a plexiglass chamber designed to hold it horizontal, a n d was continuously washed with fresh solution (Mertz, 1973). The chamber was m o u n t e d on a microscope stage, thus allowing visual observation of the insertion of t h e microelectrode into epidermal cells. P D was continuously recorded during electrode insertion a n d during long-term measurements, including periods of sample change (e.g., addition of ABA or l~aNa). All measurements were made within 30 rain from time of excision. PDs reported here are from epidermal cells; measurem e n t s are from vacuole to external solution. Electrical measurements were made using the following equipment: 3 M KCl-filled, glass micropipettes (2% agar in reference pipette); Ag/AgC1 electrodes; a n d Sargent-Welch (Chicago, IlL, USA) Model L S X pH/millivolt meter, a n d Model S R G Potentiometric Recorder. Solutions. A stock solution of 10-a M ABA was prepared b y dissolving 25 m g of dry ABA trans-ABA; Sigma Biochem. Co., St. Louis, MO., USA] in i ml of absolute ethanol, [(• a n d t h e n adding 96 ml of deionized distilled water. The p H of these solution was adjusted to 5.6 with 1 I~ KOH. The solution was stored a t 5 ~ in a n aluminium-foil-covered flask for no longer t h a n 5 days. All solutions were adjusted to p H 5.6 a n d a temperature of 24 ~ unless stated otherwise. All root b a t h i n g solutions were aerated. Most experiments utilized a two-salt solution consisting of 0.5 mM CaS04 and KC1, K~SOa, or KH2PO a. The designation KH2PO a actually represents a phosphate-buffer solution prepared b y mixing 0.1 M K2HPO a and 0.1 M KH~PO 4 in the ratio 5:95, a n d diluting to the appropriate PO a concentration (0.1 or 1 raM). 6
Planta
(Berl.), Vol. 122
D.L. Shaner et aL
82
/s
C~176/,,,"~
I
2 Time (hr)
3
4
Fig. 2. Effect of 10-5 M ABA on potassium (SeRb) accumulation from 1 mM KH~PO4+ 0.5 mM CaSO4. o = no ABA, A = 10-~ M ABA. Conditions identical to those described for Fig. 1, except S6Rb+ was included in the root bathing solution. Vertical bars = one standard deviation (if larger than symbol size}
Results 1. E]lects of A B A on Growth and Ion Uptake We have examined the effect of ABA on root growth of maize seedlings over short and long time intervals (Fig. 1A and B, respectively). 10-4 M ABA inhibited root growth b y 56% over a 25-h period {Fig. 1B). The rate of growth at each ABA level was constant from 5 to 25 h. The amount of growth between treated and control roots was significantly different (22%; P = 0.05) at both 10 -4 and 10-5 M ABA after 2 h of treatment. We have attempted to determine whether changes in salt uptake by the ABA-treated roots occurred simultaneously with or prior to the observed time of root-growth inhibition. This was studied b y monitoring the uptake of either SeRb-labeled potassium or a'P-labeled phosphate from a 1.0 mM KH2PO , solution identical to that used in the r o o t - g r o ~ h experiment. Intact seedlings were used during the period of isotope uptake. A significant inhibition (P < 0.05) of both potassium (19%) and phosphate (13%) uptake was observed within 2 h in the presence of 10-5 M ABA (Figs. 2, 3). Experiments to characterize the inhibitory effect of ABA on potassium and phosphate uptake more fully were conducted with excised low-salt roots. I n Table 1 it can be seen tha~ K + accumulation from a KC1 solution is also inhibited b y ABA. A significant inhibition (P
