Planta (Berl.) 85, 157--159 (1969)
Inhibition and Promotion by Abscisic Acid of Growth in Spirodela* * * JOHANNES VAN STADEN a n d Cm~is H. BO~NMAN Department of Botany, University of Natal, South Africa Received December 3/17, 1968
Summary. The effect of different concentrations of abscisic acid on the growth of Spirodela oligo~vhiza has been studied. Abscisie acid effectively permanently arrests growth at concentrations down to 10-1 rag/1 {one part per 10 million). Normal growth tends to be resumed at concentrations of 10-3 and 10-S rag/1 between 9 and 12 days after treatment. A concentration of 10-s rag/l, however, results in a significant increase in dry weight at both 9 and 12 days after introduction to the culture medium. It is suggested that the resumption of growth 12 days after treatment at those concentrations which inhibit growth up to 9 days, is due to a possible progressive inactivation of abscisic acid resulting in a lowering of its concentration to a level that is promotive. Sterile plants must be used since abscisie acid has shown no effect whatsoever in controlling growth of Saprolegnia, a water-mould of the Phycomycctes to which Spirodela apparently is the host. R e c e n t i n v e s t i g a t i o n on the endogenous regulator abscisic acid (ABA), have clearly d e m o n s t r a t e d its role as a m a j o r i n h i b i t o r of p l a n t growth. VAN OV]~HB~]~ et al. (1967) f o u n d t h a t Lemna minor was p a r t i c u l a r l y sensitive to A B A ; concentrations as low as one p a r t per 1,000 million (3.8 x 10 -9 M) reduced its rate of growth, while one p a r t per million (3.8 • 10-6 M) was sufficient to keep it indefinitely in a state of d o r m a n c y . This r e d u c t i o n i n growth a p p a r e n t l y is a consequency of the i n h i b i t i n g effect of A B A on nucleic acid metabolism. I n fact WAH~ING (1968) a n d V r L L I ~ S (1968) showed t h a t A B A was responsible for the i n h i b i t i o n of nucleic acid synthesis i n Taraxacum a n d Fraxinus, respectively. I n the light of abscisic acid's role i n p l a n t growth regulation, it was decided to include this substance i n a s t u d y of the effects of certain inhibitors of p r o t e i n synthesis on the growth of Spirodela oligorrhiza, a p o n d w e e d i n the family Lemnaceae. Spirodela oligorrhiza was grown in 50 m] erlenmeyer flasks in 30 ml sterile, full-strength I-Ioagland's solution, pH 4.6, with Fe-EDTA as source of iron. Each flask, containing six uniform fronds, was aerated for 45 minutes twice daily with air sterilized by passing it through a millipore filter fitted with a GS 0.22 B filter disc. Attempts to rid these plants of algae and particularly of the saprophytie fungus, Saprolegnia, proved successful only after harsh treatment by a method modified from that of GoaHA~ (1945). The plants were first submerged for four minutes in 0.1% I-IgCl~and then in 50% ethanol for 30 seconds, after which they * The authors gratefully acknowledge R . J . l%eynolds Tobacco Company, North Carolina, U.S.A., for donating the abscisic acid. ** This research was supported by a grant to the senior author from the Council for Industrial and Scientific Research, Pretoria, S.A.
158
J. v A ~ STA~E~ and Cm H. BOR~A~:
'A
\
Iscoo,,o,
,~3,
control
"~ 2 -
-9 -8 -7 -6 -5 -/. -3 -2 -1 log10 (ABA),mg/I
0
1
Fig. 1. Effect of Abscisic acid (ABA) on growth of S?irodela were transferred to sterile nutrient solution. Under these conditions there is an approximately 25% survival. Cultures were maintained at 22 ~2~ under a 16-hour photoperiod of ca. 3.0 • 103 Im/m 2. The concentrations of (RS)-abseisie acid investigated were 1O, 1 and l0 -I through 10 -9 rag/l, respectively. Each treatment consisted of six replications. Growth was measured as the increase in dry weight over either 9 or 12 days. The experiment was repeated three times. The effect of abscisic acid on Spirodela after 9 and 12 days, respectively, is shown in Fig. 1. Following treatment with 10,1, 10-1 and 10-2 mg/1 ABA growth 9 days after application was significantly inhibited as compared with tile control which had undergone an approximately five-fold increase in dry weight. ABA at 10-3 mg/1 also inhibited growth but not significantly so. These results are in general accord with those of VA~ OVERBE]~K et al. (1967) on Lemna, a genus in the same family but a considerably larger plant. However, after 12 days, the cultures treated with 10-2 and 10-3 rag/1 ABA had resumed normal growth. VA~ OV~BE]~K et al. (1967) reported a resumption of normal growth after 9 days of culturing with 100 parts per U.S. billion or less, that is, equivalent to a concentration of 10-1 mg/1 or less of ABA. Fig. 1 shows that a significant stimulation of growth was elicited in Spirodela cultured in the presence of 1 part per 100 million (10 -s mg/l) ABA. Fig. 1 furthermore indicates a promotion of growth 12 days after treatment with concentrations of lO-amg/1 ABA or less; again at 10-s mg/1 this increase in dry weight approximately 30 per cent over that
Inhibition and Promotion by Abseisic Acid
159
of the control, is significant at P = 0.05. I t is suggested that the resumption of apparently normal growth 12 days after treatment with concentration of the order of 10 -2 and 10 -s rag/1 could be due to the gradual inactivation of abscisic acid down to those concentrations low enough to promote growth. I n experiments of this nature it is imperative to use sterile plants. Spirodela is commonly infected with Saprolegnia, a phycomycetic fungus, which is not effectively killed during ordinary plant sterilization procedures. I t is of interest that none of the concentrations of ABA reported here had any inhibitory effect on the growth of this fungus; in fact, it flourished even in cultures containing 10 rag/1 ABA. (Could this apparent ineffectiveness of ABA be related to the presumably much reduced necessity of saprophytic or parasitic organisms to synthesize all their own protein requirements ?). Algae, also, appear unaffected b y abseisic acid and it is therefore essential to use sterile air when aerating the culture medium. The results and observations presented suggest that the function of abscisic acid in plant growth should not categorically be regarded as solely t h a t of inhibition although its major action probably is analogous to that of a plant drug. I t does appear to have promotive effects (Aspt1gALL et al., 1967); such as in the acceleration of abscission (BoR~MAS et al., 1967) where the synthesis of certain degradative enzymes of the pectic constituents of the cell wall is probably stimulated by ABA. The present authors feel that further evidence has been presented in support of VAN OV~Br~X'S (1968) and VInLIERS' (1968) use of the term hormone in reference to abscisic acid. References ASPINALL, D., L. G. PALEO, and F. T. ADDICOTT: Abscisin I I and some hormone regulated plant responses. Aust. J. biol. Sci. 20, 869--882 (1967). BOR~MA~, C. H., A. t~. SPuRR, and F. T. ADDICOT~r: Abscisin, auxin, and gibberellin effects on the developmental aspects of abscission in cotton (Gossypium hirsuture). Amer. J. Bot. $4, 125--135 (1967). GO~AM, P . R . : Growth factor studies with Spirodela polyrrhiza (L) Schleid. Amer. J. Bot. 32, 496--505 (1945). O V E ~ ] ~ : , J. vA~: The control of plant growth. Sci. Amer. 75--81 (July, 1968). - - J. E. LOnFFL~R, and M. I. t~. MASOn: Dormin (Abseisin II), inhibitor of plant DNA synthesis. Science 156, 1497--1499 (1967). VILLIE~S, T. A. : An autoradiographic study of the effect of the plant hormone abscisic acid on nucleic acid and protein metabolism. Planta (Berl.) 82, 342--354 (1968). WA~EING, P. F. : The possible role and mode of action of abscisic acid in plant growth regulation. Abstr. Society of Chemical Industry Symposium on Plant Growth Regulators, London, 1968. Mr. J. VAN STAD~ and Professor C. H. BOR~MA~ Department of Botany, University of Natal Pietermaritzburg, Natal, l~epublic of South Africa 11 Planta (Berl.), Bd. 85
Inhibition and Promotion by Abscisic Acid of Growth in Spirodela* * * JOHANNES VAN STADEN a n d Cm~is H. BO~NMAN Department of Botany, University of Natal, South Africa Received December 3/17, 1968
Summary. The effect of different concentrations of abscisic acid on the growth of Spirodela oligo~vhiza has been studied. Abscisie acid effectively permanently arrests growth at concentrations down to 10-1 rag/1 {one part per 10 million). Normal growth tends to be resumed at concentrations of 10-3 and 10-S rag/1 between 9 and 12 days after treatment. A concentration of 10-s rag/l, however, results in a significant increase in dry weight at both 9 and 12 days after introduction to the culture medium. It is suggested that the resumption of growth 12 days after treatment at those concentrations which inhibit growth up to 9 days, is due to a possible progressive inactivation of abscisic acid resulting in a lowering of its concentration to a level that is promotive. Sterile plants must be used since abscisie acid has shown no effect whatsoever in controlling growth of Saprolegnia, a water-mould of the Phycomycctes to which Spirodela apparently is the host. R e c e n t i n v e s t i g a t i o n on the endogenous regulator abscisic acid (ABA), have clearly d e m o n s t r a t e d its role as a m a j o r i n h i b i t o r of p l a n t growth. VAN OV]~HB~]~ et al. (1967) f o u n d t h a t Lemna minor was p a r t i c u l a r l y sensitive to A B A ; concentrations as low as one p a r t per 1,000 million (3.8 x 10 -9 M) reduced its rate of growth, while one p a r t per million (3.8 • 10-6 M) was sufficient to keep it indefinitely in a state of d o r m a n c y . This r e d u c t i o n i n growth a p p a r e n t l y is a consequency of the i n h i b i t i n g effect of A B A on nucleic acid metabolism. I n fact WAH~ING (1968) a n d V r L L I ~ S (1968) showed t h a t A B A was responsible for the i n h i b i t i o n of nucleic acid synthesis i n Taraxacum a n d Fraxinus, respectively. I n the light of abscisic acid's role i n p l a n t growth regulation, it was decided to include this substance i n a s t u d y of the effects of certain inhibitors of p r o t e i n synthesis on the growth of Spirodela oligorrhiza, a p o n d w e e d i n the family Lemnaceae. Spirodela oligorrhiza was grown in 50 m] erlenmeyer flasks in 30 ml sterile, full-strength I-Ioagland's solution, pH 4.6, with Fe-EDTA as source of iron. Each flask, containing six uniform fronds, was aerated for 45 minutes twice daily with air sterilized by passing it through a millipore filter fitted with a GS 0.22 B filter disc. Attempts to rid these plants of algae and particularly of the saprophytie fungus, Saprolegnia, proved successful only after harsh treatment by a method modified from that of GoaHA~ (1945). The plants were first submerged for four minutes in 0.1% I-IgCl~and then in 50% ethanol for 30 seconds, after which they * The authors gratefully acknowledge R . J . l%eynolds Tobacco Company, North Carolina, U.S.A., for donating the abscisic acid. ** This research was supported by a grant to the senior author from the Council for Industrial and Scientific Research, Pretoria, S.A.
158
J. v A ~ STA~E~ and Cm H. BOR~A~:
'A
\
Iscoo,,o,
,~3,
control
"~ 2 -
-9 -8 -7 -6 -5 -/. -3 -2 -1 log10 (ABA),mg/I
0
1
Fig. 1. Effect of Abscisic acid (ABA) on growth of S?irodela were transferred to sterile nutrient solution. Under these conditions there is an approximately 25% survival. Cultures were maintained at 22 ~2~ under a 16-hour photoperiod of ca. 3.0 • 103 Im/m 2. The concentrations of (RS)-abseisie acid investigated were 1O, 1 and l0 -I through 10 -9 rag/l, respectively. Each treatment consisted of six replications. Growth was measured as the increase in dry weight over either 9 or 12 days. The experiment was repeated three times. The effect of abscisic acid on Spirodela after 9 and 12 days, respectively, is shown in Fig. 1. Following treatment with 10,1, 10-1 and 10-2 mg/1 ABA growth 9 days after application was significantly inhibited as compared with tile control which had undergone an approximately five-fold increase in dry weight. ABA at 10-3 mg/1 also inhibited growth but not significantly so. These results are in general accord with those of VA~ OVERBE]~K et al. (1967) on Lemna, a genus in the same family but a considerably larger plant. However, after 12 days, the cultures treated with 10-2 and 10-3 rag/1 ABA had resumed normal growth. VA~ OV~BE]~K et al. (1967) reported a resumption of normal growth after 9 days of culturing with 100 parts per U.S. billion or less, that is, equivalent to a concentration of 10-1 mg/1 or less of ABA. Fig. 1 shows that a significant stimulation of growth was elicited in Spirodela cultured in the presence of 1 part per 100 million (10 -s mg/l) ABA. Fig. 1 furthermore indicates a promotion of growth 12 days after treatment with concentrations of lO-amg/1 ABA or less; again at 10-s mg/1 this increase in dry weight approximately 30 per cent over that
Inhibition and Promotion by Abseisic Acid
159
of the control, is significant at P = 0.05. I t is suggested that the resumption of apparently normal growth 12 days after treatment with concentration of the order of 10 -2 and 10 -s rag/1 could be due to the gradual inactivation of abscisic acid down to those concentrations low enough to promote growth. I n experiments of this nature it is imperative to use sterile plants. Spirodela is commonly infected with Saprolegnia, a phycomycetic fungus, which is not effectively killed during ordinary plant sterilization procedures. I t is of interest that none of the concentrations of ABA reported here had any inhibitory effect on the growth of this fungus; in fact, it flourished even in cultures containing 10 rag/1 ABA. (Could this apparent ineffectiveness of ABA be related to the presumably much reduced necessity of saprophytic or parasitic organisms to synthesize all their own protein requirements ?). Algae, also, appear unaffected b y abseisic acid and it is therefore essential to use sterile air when aerating the culture medium. The results and observations presented suggest that the function of abscisic acid in plant growth should not categorically be regarded as solely t h a t of inhibition although its major action probably is analogous to that of a plant drug. I t does appear to have promotive effects (Aspt1gALL et al., 1967); such as in the acceleration of abscission (BoR~MAS et al., 1967) where the synthesis of certain degradative enzymes of the pectic constituents of the cell wall is probably stimulated by ABA. The present authors feel that further evidence has been presented in support of VAN OV~Br~X'S (1968) and VInLIERS' (1968) use of the term hormone in reference to abscisic acid. References ASPINALL, D., L. G. PALEO, and F. T. ADDICOTT: Abscisin I I and some hormone regulated plant responses. Aust. J. biol. Sci. 20, 869--882 (1967). BOR~MA~, C. H., A. t~. SPuRR, and F. T. ADDICOT~r: Abscisin, auxin, and gibberellin effects on the developmental aspects of abscission in cotton (Gossypium hirsuture). Amer. J. Bot. $4, 125--135 (1967). GO~AM, P . R . : Growth factor studies with Spirodela polyrrhiza (L) Schleid. Amer. J. Bot. 32, 496--505 (1945). O V E ~ ] ~ : , J. vA~: The control of plant growth. Sci. Amer. 75--81 (July, 1968). - - J. E. LOnFFL~R, and M. I. t~. MASOn: Dormin (Abseisin II), inhibitor of plant DNA synthesis. Science 156, 1497--1499 (1967). VILLIE~S, T. A. : An autoradiographic study of the effect of the plant hormone abscisic acid on nucleic acid and protein metabolism. Planta (Berl.) 82, 342--354 (1968). WA~EING, P. F. : The possible role and mode of action of abscisic acid in plant growth regulation. Abstr. Society of Chemical Industry Symposium on Plant Growth Regulators, London, 1968. Mr. J. VAN STAD~ and Professor C. H. BOR~MA~ Department of Botany, University of Natal Pietermaritzburg, Natal, l~epublic of South Africa 11 Planta (Berl.), Bd. 85
