Planta (Berl.) 104, 182-184 (i972) 9 by Springer-Verlag 1972
Short Communication Abscisic Acid in Pea Shoots M. !~. B a r n e s Biochemistry Department, Lincoln College, Canterbury, New Zealand Received December 10, 1971
Summary. The (~)-abscisic acid content of pea shoots has been determined using gas-liquid chromatography. In both tall and dwarf cultivars no significant difference was observed between plants grown in the dark or under red light. Nor was the difference between the tall and dwarf cultivars themselves significant. The p l a n t g r o w t h i n h i b i t i n g substance (~-)-abscisic a c i d (ABA) is k n o w n to h a v e a w i d e s p r e a d d i s t r i b u t i o n t h r o u g h o u t t h e p l a n t k i n g d o m ( A d d i c o t t a n d L y o n , 1969). A s a r e s u l t of earlier w o r k w i t h ~his s u b s t a n c e i t seemed to t h e a u t h o r t h a t d w a r f i s m in p l a n t s m a y be a n expression of t h e c o n t e n t of A B A in t h e p l a n t tissue. P r e l i m i n a r y e x p e r i m e n t s (Barnes a n d L i g h t , 1969) a p p e a r e d to i n d i c a t e a higher A B A c o n t e n t in d w a r f t h a n in t a l l peas, a n d t h u s to suggest a direct role in dwarfism. R e c e n t l y , however, more extensive e x p e r i m e n t s h a v e failed to confirm this result a n d p r o m p t this brief r e p o r t on t h e c o n t i n u e d work. The ABA content of peas (Pisum sativum L.) was determined by a modification of the extraction and gas-liquid chromatographic (GLC) procedure used previously (Barnes and Light, 1969), The peas, a tall (Gradus) and a dwarf cultivar (Progress No. 9), were grown in a controlled-environment chamber at 25 ~ C, either without light or with continuous red light. The light source was two 100-W bulbs fitted with red filters, approximately 25 cm above the plants. After 10 days the shoo~s (800 g) were excised, homogenised in 95% methanol, and the resulting slurry was stirred at 3~ for 16 h in the dark. After filtration the extraction was repeated with the residue. The combined filtrates were evaporated at 40~ and the residue was dissolved in 5% aqueous sodium bicarbonate (10 ml), acidified to pH 2.5, and the ABA extracted into diethyl ether (2 x 50 ml). The crude ABA in this ether extract was purified by thin-layer chromatography (TLC) on Silica gel G (Merck), first in the solvent mixture: n-butanol, 2; n-propanol, 6; 0.88 ammonia, 1; water, 2; and second in benzene, 100; ethyl acetate, 50; acetic acid, 2. The ultraviolet-absorbing band, chromatographing with an authentic sample of ABA, was eluted in each case with ethyl acetate. Quantitation by GLC was carried out in a Perkin-Elmer F-11 twin-column instrument with a flame ionisation detector. The glass column (ca. 90 c m x 6.5 ram)
Abscisic Acid in Pea Shoots
183
was packed with 0.5% QF-1 (fluorinated silicone oil) on Chromosorb W (diatomaceous earth, Johns-Manville Products Corp.). Temperature programming at 4~ from 130 to 180~ was used. Table 1 shows clearly that the variation of ABA content within the pea shoot is not significant in relation to the changes in shoot length brought about by exposure to red light. Also the result that the ABA content of the dwarf cultivar (Progress No. 9), if anything, is slightly less than the ABA content of the tall cultivar (Gradus) discounts the possibility that differences in ABA content arc involved in the control of the height of the shoot. Table 1. The ABA content of tall and dwarf peas grown under two light regimes Expt.
Cultivar
Light treatment
ABA content
Height of plants (cm)
~g/plant:
~g/kg fr.wt,
~g/kg dry wt.
Gradus
Dark Red light
0.032 0.030
43 38
1020 870
38-43 21-24
Progress No. 9
Dark Red light
0.027 0.023
22 33
820 700
23-26 6-8
Gradus
Dark Red light
0.014 0.016
17 33
860 630
22-26 8-12
Progress No. 9
Dark Red light
0.005 0.009
54 23
151 360
15-18 6-8
Gradus
Dark Red light
0.016 0.021
18 37
---
28-33 15-19
Progress No. 9
Dark Red light
0.009 0.015
12 38
---
16-20 6-8
The differences between the figures reported here and the prellmiuary data mentioned in Barnes and Light (1969) require some explanation. I t is b y now well known that wilting causes a marked increase in the ABA content of plant tissue (Wright and Hiron, 1969; Zeevart, 1971; Simpson and Saunders, 1972). I t is unlikely however that wilting was responsible for the original, erroneous result since the seedlings were kept well watered and free from harsh air disturbances. A more likely explanation is that the random losses in the lengthy extraction procedure were greater than the ABA content of the small amount of pea tissue used (ca. 25 g fresh weight) in the exploratory investigation. The results given in Table 1 are in accord with those given recently by other workers (Kende and Kays, 1971 ; Simpson and Saunders, 1972).
184
M.F. Barnes: Abseisie Acid in Pea Shoots
There are however some slight differences in the figures reported in t h a t while Kende and K a y s indicate no significant difference between the A B A content of dwarf peas grown in the light or in the dark (1.5 and 1.7 ~g/1000 seedlings), the figures are lower t h a n those reported here b y a factor of 10. On the other h a n d the figures reported b y Simpson and Saunders are in agreement with mine in terms of magnitude, while again indicating no significant difference in A B A content of the various tissues. I t is now quite clear from the data available t h a t the actual content of A B A in pea tissue is not a factor in the control of elongation of the shoot. The author wishes to thank Dr. R. C. Peeket, Botany Department, Victoria University, Manchester for making available the growth cabinet facilities, Mr. P./-Iflling for technical assistance and F. Hoffmann-La Roche & Co., Ltd., Basel, Switzerland for a sample of authentic (• acid.
References Addicott, F. T., Lyon, J. L.: Physiology of abscisie acid and related substances. Ann. Rev. Plant. Physiol. 20, 139-164 (1969). Barnes, M. F., Light, E. N. : Occurrence of abseisic acid in the gibberellin--inhibi~r from limabeans. Planta (Berl.) 89, 303-308 (1969). Kende, H., Kays, S. E. : The level of (-~)-abscisic acid in dwarf pea shoots. Naturwissenschaften 58, 524-525 (1971). Simpson, G.M., Saunders, P. F.: Abscisie acid associated with wilting in dwarf and tall P i s u m sativum. Planta (Berl.) 102, 272-276 (1972). Wright, S. T. C., Hiron, R. W. P. : (~)-Abseisic acid, the growth inhibitor induced in detached wheat leaves by a period of wilting. Nature (Lond.) 224, 719-720 (1969). Zeevart, J. A. D. : (-}-)-Abscisic acid content of spinach in relation to photoperiod and water stress. Plant Physiol. 48, 86-90 (1971). M. F. Barnes Biochemistry Department Lincoln College Canterbury, New Zealand
Short Communication Abscisic Acid in Pea Shoots M. !~. B a r n e s Biochemistry Department, Lincoln College, Canterbury, New Zealand Received December 10, 1971
Summary. The (~)-abscisic acid content of pea shoots has been determined using gas-liquid chromatography. In both tall and dwarf cultivars no significant difference was observed between plants grown in the dark or under red light. Nor was the difference between the tall and dwarf cultivars themselves significant. The p l a n t g r o w t h i n h i b i t i n g substance (~-)-abscisic a c i d (ABA) is k n o w n to h a v e a w i d e s p r e a d d i s t r i b u t i o n t h r o u g h o u t t h e p l a n t k i n g d o m ( A d d i c o t t a n d L y o n , 1969). A s a r e s u l t of earlier w o r k w i t h ~his s u b s t a n c e i t seemed to t h e a u t h o r t h a t d w a r f i s m in p l a n t s m a y be a n expression of t h e c o n t e n t of A B A in t h e p l a n t tissue. P r e l i m i n a r y e x p e r i m e n t s (Barnes a n d L i g h t , 1969) a p p e a r e d to i n d i c a t e a higher A B A c o n t e n t in d w a r f t h a n in t a l l peas, a n d t h u s to suggest a direct role in dwarfism. R e c e n t l y , however, more extensive e x p e r i m e n t s h a v e failed to confirm this result a n d p r o m p t this brief r e p o r t on t h e c o n t i n u e d work. The ABA content of peas (Pisum sativum L.) was determined by a modification of the extraction and gas-liquid chromatographic (GLC) procedure used previously (Barnes and Light, 1969), The peas, a tall (Gradus) and a dwarf cultivar (Progress No. 9), were grown in a controlled-environment chamber at 25 ~ C, either without light or with continuous red light. The light source was two 100-W bulbs fitted with red filters, approximately 25 cm above the plants. After 10 days the shoo~s (800 g) were excised, homogenised in 95% methanol, and the resulting slurry was stirred at 3~ for 16 h in the dark. After filtration the extraction was repeated with the residue. The combined filtrates were evaporated at 40~ and the residue was dissolved in 5% aqueous sodium bicarbonate (10 ml), acidified to pH 2.5, and the ABA extracted into diethyl ether (2 x 50 ml). The crude ABA in this ether extract was purified by thin-layer chromatography (TLC) on Silica gel G (Merck), first in the solvent mixture: n-butanol, 2; n-propanol, 6; 0.88 ammonia, 1; water, 2; and second in benzene, 100; ethyl acetate, 50; acetic acid, 2. The ultraviolet-absorbing band, chromatographing with an authentic sample of ABA, was eluted in each case with ethyl acetate. Quantitation by GLC was carried out in a Perkin-Elmer F-11 twin-column instrument with a flame ionisation detector. The glass column (ca. 90 c m x 6.5 ram)
Abscisic Acid in Pea Shoots
183
was packed with 0.5% QF-1 (fluorinated silicone oil) on Chromosorb W (diatomaceous earth, Johns-Manville Products Corp.). Temperature programming at 4~ from 130 to 180~ was used. Table 1 shows clearly that the variation of ABA content within the pea shoot is not significant in relation to the changes in shoot length brought about by exposure to red light. Also the result that the ABA content of the dwarf cultivar (Progress No. 9), if anything, is slightly less than the ABA content of the tall cultivar (Gradus) discounts the possibility that differences in ABA content arc involved in the control of the height of the shoot. Table 1. The ABA content of tall and dwarf peas grown under two light regimes Expt.
Cultivar
Light treatment
ABA content
Height of plants (cm)
~g/plant:
~g/kg fr.wt,
~g/kg dry wt.
Gradus
Dark Red light
0.032 0.030
43 38
1020 870
38-43 21-24
Progress No. 9
Dark Red light
0.027 0.023
22 33
820 700
23-26 6-8
Gradus
Dark Red light
0.014 0.016
17 33
860 630
22-26 8-12
Progress No. 9
Dark Red light
0.005 0.009
54 23
151 360
15-18 6-8
Gradus
Dark Red light
0.016 0.021
18 37
---
28-33 15-19
Progress No. 9
Dark Red light
0.009 0.015
12 38
---
16-20 6-8
The differences between the figures reported here and the prellmiuary data mentioned in Barnes and Light (1969) require some explanation. I t is b y now well known that wilting causes a marked increase in the ABA content of plant tissue (Wright and Hiron, 1969; Zeevart, 1971; Simpson and Saunders, 1972). I t is unlikely however that wilting was responsible for the original, erroneous result since the seedlings were kept well watered and free from harsh air disturbances. A more likely explanation is that the random losses in the lengthy extraction procedure were greater than the ABA content of the small amount of pea tissue used (ca. 25 g fresh weight) in the exploratory investigation. The results given in Table 1 are in accord with those given recently by other workers (Kende and Kays, 1971 ; Simpson and Saunders, 1972).
184
M.F. Barnes: Abseisie Acid in Pea Shoots
There are however some slight differences in the figures reported in t h a t while Kende and K a y s indicate no significant difference between the A B A content of dwarf peas grown in the light or in the dark (1.5 and 1.7 ~g/1000 seedlings), the figures are lower t h a n those reported here b y a factor of 10. On the other h a n d the figures reported b y Simpson and Saunders are in agreement with mine in terms of magnitude, while again indicating no significant difference in A B A content of the various tissues. I t is now quite clear from the data available t h a t the actual content of A B A in pea tissue is not a factor in the control of elongation of the shoot. The author wishes to thank Dr. R. C. Peeket, Botany Department, Victoria University, Manchester for making available the growth cabinet facilities, Mr. P./-Iflling for technical assistance and F. Hoffmann-La Roche & Co., Ltd., Basel, Switzerland for a sample of authentic (• acid.
References Addicott, F. T., Lyon, J. L.: Physiology of abscisie acid and related substances. Ann. Rev. Plant. Physiol. 20, 139-164 (1969). Barnes, M. F., Light, E. N. : Occurrence of abseisic acid in the gibberellin--inhibi~r from limabeans. Planta (Berl.) 89, 303-308 (1969). Kende, H., Kays, S. E. : The level of (-~)-abscisic acid in dwarf pea shoots. Naturwissenschaften 58, 524-525 (1971). Simpson, G.M., Saunders, P. F.: Abscisie acid associated with wilting in dwarf and tall P i s u m sativum. Planta (Berl.) 102, 272-276 (1972). Wright, S. T. C., Hiron, R. W. P. : (~)-Abseisic acid, the growth inhibitor induced in detached wheat leaves by a period of wilting. Nature (Lond.) 224, 719-720 (1969). Zeevart, J. A. D. : (-}-)-Abscisic acid content of spinach in relation to photoperiod and water stress. Plant Physiol. 48, 86-90 (1971). M. F. Barnes Biochemistry Department Lincoln College Canterbury, New Zealand
