Planta
Planta 150; 349-353 (1980)
9 by Springer-Verlag 1980
Stimulation by Abscisic Acid of RNA Synthesis in Discs from Healthy and Tobacco Mosaic Virus-infected Tobacco Leaves R.J. W h e n h a m a n d R.S.S. F r a s e r Biochemistry Section, National Vegetable Research Station, Wellesbourne, Warwick CV35 9EF, U.K.
Abstract. U p t a k e o f abscisic acid f r o m the c u l t u r e m e d i u m by discs o f h e a l t h y a n d t o b a c c o m o s a i c virusinfected t o b a c c o leaves was m e a s u r e d . Small (two to five-fold) increases in abscisic acid c o n c e n t r a t i o n in discs c a u s e d increases in rates o f [3H]uridine a n d [3H]adenine i n c o r p o r a t i o n into t o t a l nucleic acid, virus R N A a n d h o s t r i b o s o m a l R N A . N e t a c c u m u l a tion o f virus R N A was also e n h a n c e d by abscisic acid. This evidence for s t i m u l a t i o n o f R N A synthesis is c o m p a r e d with p r e v i o u s r e p o r t s s h o w i n g i n h i b i t i o n o f R N A synthesis in o t h e r tissues. It is suggested t h a t the increase in e n d o g e n o u s abscisic acid caused by t o b a c c o m o s a i c virus infection m a y be at least p a r t l y r e s p o n s i b l e for o b s e r v e d increases in rates o f R N A synthesis after infection. Key words: A b s c i s i c acid - N i c o t i a n a - R i b o n u c l e i c acid synthesis - T o b a c c o m o s a i c virus.
Introduction W h e n abscisic acid ( A B A ) is a p p l i e d to p l a n t tissues, its r e p o r t e d effects i n c l u d e i n h i b i t i o n o f g r o w t h (reviewed by M i l b o r r o w 1974) a n d R N A synthesis (Villiers 1968; Pilet 1970; Bex 1972a; Z w a r a n d J a c o b s e n 1972; W a l b o t e t a l . 1975; M i n o c h a a n d D i B o n a 1979). T h e m e c h a n i s m o f i n h i b i t i o n o f R N A synthesis is n o t u n d e r s t o o d : it has been suggested t h a t A B A m a y indirectly interfere with c h r o m a t i n activity (Pearson a n d W a r e i n g 1969) a n d R N A p o l y m e r a s e activity (Bex 1972b). I n f e c t i o n o f t o b a c c o leaves b y t o b a c c o m o s a i c virus ( T M V ) increases their e n d o g e n o u s A B A c o n c e n t r a t i o n b y t w o to five fold (R.J. W h e n h a m a n d R.S.S. F r a s e r , s u b m i t t e d m a n u s c r i p t ) . R N A synthesis in virus-infected leaves is at a high r a t e : T M V Abbreviations: ABA=abscisic acid; TMV=tobacco mosaic virus
R N A a c c u m u l a t e s to as m u c h as 500 gg p e r g fresh weight, a n d h o s t r i b o s o m a l R N A synthesis is stimulated in the early stages o f infection ( F r a s e r 1972, 1973). These findings p r o m p t e d us to e x a m i n e the effects o f i n c r e a s e d A B A c o n c e n t r a t i o n on R N A synthesis in t o b a c c o leaves. O u r results suggest t h a t in this tissue, small increases in A B A c o n c e n t r a t i o n m a y s t i m u l a t e R N A synthesis a n d a c c u m u l a t i o n .
Materials and Methods Tobacco plants (Nicotiana tabacum L. cvs Samsun and White Burley) were grown and inoculated on two expanded lower leaves with TMV strain vulgare as described by Fraser and Whenham (1978). Upper leaves became infected by systemic spread of virus. Those which had expanded to about one-half final size were harvested one day after appearance of mosaic symptoms, i.e. very early in the virus multiplication phase (Fraser 1972). Comparable leaves were also taken from healthy plants. After surface sterilization for 15 min in 0.01% benzalkonium chloride; 5% ethanol, leaves were washed three times with sterile water and discs of 6 or 15 mm diameter removed by cork borer. Groups of four discs were floated on sterile B5 medium (Gamborg et al. 1968) containing various concentrations of (_+) cis-trans-abscisic acid (Sigma Chemical Co. UK) but no other hormones. Discs were infiltrated under vacuum for 2 rain then incubated in a growth cabinet at 25~ with an irradiance of 50 W m -2 and a 14 h daylength. Each treatment was replicated three or five times. [5-3H]uridine (specific activity 0.9 TBq mmo1-1) or [2-3H]adenine (specific activity 0.75 TBq mmol 1) (Radiochemical Centre, Amersham USA) were added to label nucleic acids via the pyrimidine or purine precursor pathways respectively. Radioactive concentrations and time of labelling are given in results. For measurement of total uptake and incorporation of radioactivity, each group of four discs was rinsed in water and lightly blotted, then homogenized in 1 ml 0.5 M HC104 at 0~ C. The homogenate was centrifuged at 12,000 g for 3 rain, and acid-soluble radioactivity measured by counting 0.1 ml of supernatant in 8 ml scintillation liquid (0.5% 2-(4"-tert-butylphenyl)-5-(4"-biphenylyl)1,3,4-oxadiazole in toluene: 2-methoxyethanol 6:4 by volume). Correction of counts to 100% counting efficiency was by the external standard channels ratio method. The precipitate was suspended in 1 ml 0.5 M HC104, collected by filtration on a Whatman GF/A glass fibre disc and washed
0032-0935/80/0150/0349/$01.00
350
three times with 4 ml 0.5 M HC104. Nucleic acids on the filter were hydrolyzed at 70~ for 30 min with 0.3 ml 0.5 M HC104, then cold acid-insoluble radioactivity (incorporation) was determined by counting as above. Total isotope uptake was acid-soluble plus acid-insoluble radioactivity. Amounts and rates of synthesis of individual R N A species were measured by extracting total nucleic acids by a phenol-detergent procedure as described by Fraser and Whenham (1978). The nucleic acids were fractionated by electrophoresis on 2.1% polyacrylamide gels (Loening 1969) for 3 h at 5 mA/gel, 8 V cm 1. Gels were scanned for absorption at 265 nm and the amount of TMV RNA determined from the area of its ultraviolet absorption peak (Fraser 1971). Gels were cut into 1 mm thick slices and radioactivity in each slice measured by HC104 hydrolysis and scintillation counting as above. ABA concentrations in healthy and infected leaf tissues were measured by extraction and gas-liquid chromatography as described by Fraser et al. (1979). ABA uptake by discs was measured by floating them on B 5 medium containing various concentrations of total A BA and known radioactivities of ( + ) cis-trans-[2-l~C]abscisic acid (Radiochemical Centre, Amersham; specific activity 440 MBq mmol-1). After incubation, the discs were rinsed in water, lightly blotted and homogenized in 80% methanol at 0 ~ C. Methanol was removed under vacuum and [~4C]ABA separated into free acid and ' b o u n d ' fractions (the latter presumed to be the glucosyl ester; Milborrow (1970)), by acid/base partitioning of the homogenate with dichloromethane (Zabadal 1974). Radioactivity in free acid and ' b o u n d ' ABA fractions was measured by scintillation counting as above.
Results
ABA Uptake. In a typical experiment, the endogenous ABA concentration of healthy tobacco leaves was 24_+2 ng g 1 fresh weight (mean + standard error). Comparable, TMV-infected leaves contained 4 6 + 6 ng g-1. Endogenous ABA concentration varied with leaf age and environmental conditions: over all experiments the range was from 10-40ng g t in healthy leaves and 20-80 ng g a in TMV-infected leaves. Infected leaves consistently contained between two and five times the ABA concentration of comparable healthy leaves sampled at the same time. Leaf discs took up ABA from the culture medium for at least 24 h at a constant rate. After 24 h exposure to [I~C]ABA, 40% of the ABA taken up was recovered as the free acid and 60% was as the presumed glucosyl ester form, considered to be the major rapidstorage form of ABA (Milborrow 1970). This ratio of free to bound ABA was independent of exogenous ABA concentration. Uptake of ABA varied somewhat from experiment to experiment; overall it depended strongly on ABA concentration in the medium over a very wide range (Fig. 1). Discs from TMV-infected leaves consistently took up more ABA than comparable discs from healthy leaves: over all experiments ABA uptake by infected discs was 3.3_+ 0.3 times (mean + S.E.) that in healthy discs.
R.J. W h e n h a m and R.S.S. Fraser: A B A and R N A Synthesis
•'•10
e
10 ~
./
.-~,~104 103
./S ~
,~%~10=
Planta 150; 349-353 (1980)
9 by Springer-Verlag 1980
Stimulation by Abscisic Acid of RNA Synthesis in Discs from Healthy and Tobacco Mosaic Virus-infected Tobacco Leaves R.J. W h e n h a m a n d R.S.S. F r a s e r Biochemistry Section, National Vegetable Research Station, Wellesbourne, Warwick CV35 9EF, U.K.
Abstract. U p t a k e o f abscisic acid f r o m the c u l t u r e m e d i u m by discs o f h e a l t h y a n d t o b a c c o m o s a i c virusinfected t o b a c c o leaves was m e a s u r e d . Small (two to five-fold) increases in abscisic acid c o n c e n t r a t i o n in discs c a u s e d increases in rates o f [3H]uridine a n d [3H]adenine i n c o r p o r a t i o n into t o t a l nucleic acid, virus R N A a n d h o s t r i b o s o m a l R N A . N e t a c c u m u l a tion o f virus R N A was also e n h a n c e d by abscisic acid. This evidence for s t i m u l a t i o n o f R N A synthesis is c o m p a r e d with p r e v i o u s r e p o r t s s h o w i n g i n h i b i t i o n o f R N A synthesis in o t h e r tissues. It is suggested t h a t the increase in e n d o g e n o u s abscisic acid caused by t o b a c c o m o s a i c virus infection m a y be at least p a r t l y r e s p o n s i b l e for o b s e r v e d increases in rates o f R N A synthesis after infection. Key words: A b s c i s i c acid - N i c o t i a n a - R i b o n u c l e i c acid synthesis - T o b a c c o m o s a i c virus.
Introduction W h e n abscisic acid ( A B A ) is a p p l i e d to p l a n t tissues, its r e p o r t e d effects i n c l u d e i n h i b i t i o n o f g r o w t h (reviewed by M i l b o r r o w 1974) a n d R N A synthesis (Villiers 1968; Pilet 1970; Bex 1972a; Z w a r a n d J a c o b s e n 1972; W a l b o t e t a l . 1975; M i n o c h a a n d D i B o n a 1979). T h e m e c h a n i s m o f i n h i b i t i o n o f R N A synthesis is n o t u n d e r s t o o d : it has been suggested t h a t A B A m a y indirectly interfere with c h r o m a t i n activity (Pearson a n d W a r e i n g 1969) a n d R N A p o l y m e r a s e activity (Bex 1972b). I n f e c t i o n o f t o b a c c o leaves b y t o b a c c o m o s a i c virus ( T M V ) increases their e n d o g e n o u s A B A c o n c e n t r a t i o n b y t w o to five fold (R.J. W h e n h a m a n d R.S.S. F r a s e r , s u b m i t t e d m a n u s c r i p t ) . R N A synthesis in virus-infected leaves is at a high r a t e : T M V Abbreviations: ABA=abscisic acid; TMV=tobacco mosaic virus
R N A a c c u m u l a t e s to as m u c h as 500 gg p e r g fresh weight, a n d h o s t r i b o s o m a l R N A synthesis is stimulated in the early stages o f infection ( F r a s e r 1972, 1973). These findings p r o m p t e d us to e x a m i n e the effects o f i n c r e a s e d A B A c o n c e n t r a t i o n on R N A synthesis in t o b a c c o leaves. O u r results suggest t h a t in this tissue, small increases in A B A c o n c e n t r a t i o n m a y s t i m u l a t e R N A synthesis a n d a c c u m u l a t i o n .
Materials and Methods Tobacco plants (Nicotiana tabacum L. cvs Samsun and White Burley) were grown and inoculated on two expanded lower leaves with TMV strain vulgare as described by Fraser and Whenham (1978). Upper leaves became infected by systemic spread of virus. Those which had expanded to about one-half final size were harvested one day after appearance of mosaic symptoms, i.e. very early in the virus multiplication phase (Fraser 1972). Comparable leaves were also taken from healthy plants. After surface sterilization for 15 min in 0.01% benzalkonium chloride; 5% ethanol, leaves were washed three times with sterile water and discs of 6 or 15 mm diameter removed by cork borer. Groups of four discs were floated on sterile B5 medium (Gamborg et al. 1968) containing various concentrations of (_+) cis-trans-abscisic acid (Sigma Chemical Co. UK) but no other hormones. Discs were infiltrated under vacuum for 2 rain then incubated in a growth cabinet at 25~ with an irradiance of 50 W m -2 and a 14 h daylength. Each treatment was replicated three or five times. [5-3H]uridine (specific activity 0.9 TBq mmo1-1) or [2-3H]adenine (specific activity 0.75 TBq mmol 1) (Radiochemical Centre, Amersham USA) were added to label nucleic acids via the pyrimidine or purine precursor pathways respectively. Radioactive concentrations and time of labelling are given in results. For measurement of total uptake and incorporation of radioactivity, each group of four discs was rinsed in water and lightly blotted, then homogenized in 1 ml 0.5 M HC104 at 0~ C. The homogenate was centrifuged at 12,000 g for 3 rain, and acid-soluble radioactivity measured by counting 0.1 ml of supernatant in 8 ml scintillation liquid (0.5% 2-(4"-tert-butylphenyl)-5-(4"-biphenylyl)1,3,4-oxadiazole in toluene: 2-methoxyethanol 6:4 by volume). Correction of counts to 100% counting efficiency was by the external standard channels ratio method. The precipitate was suspended in 1 ml 0.5 M HC104, collected by filtration on a Whatman GF/A glass fibre disc and washed
0032-0935/80/0150/0349/$01.00
350
three times with 4 ml 0.5 M HC104. Nucleic acids on the filter were hydrolyzed at 70~ for 30 min with 0.3 ml 0.5 M HC104, then cold acid-insoluble radioactivity (incorporation) was determined by counting as above. Total isotope uptake was acid-soluble plus acid-insoluble radioactivity. Amounts and rates of synthesis of individual R N A species were measured by extracting total nucleic acids by a phenol-detergent procedure as described by Fraser and Whenham (1978). The nucleic acids were fractionated by electrophoresis on 2.1% polyacrylamide gels (Loening 1969) for 3 h at 5 mA/gel, 8 V cm 1. Gels were scanned for absorption at 265 nm and the amount of TMV RNA determined from the area of its ultraviolet absorption peak (Fraser 1971). Gels were cut into 1 mm thick slices and radioactivity in each slice measured by HC104 hydrolysis and scintillation counting as above. ABA concentrations in healthy and infected leaf tissues were measured by extraction and gas-liquid chromatography as described by Fraser et al. (1979). ABA uptake by discs was measured by floating them on B 5 medium containing various concentrations of total A BA and known radioactivities of ( + ) cis-trans-[2-l~C]abscisic acid (Radiochemical Centre, Amersham; specific activity 440 MBq mmol-1). After incubation, the discs were rinsed in water, lightly blotted and homogenized in 80% methanol at 0 ~ C. Methanol was removed under vacuum and [~4C]ABA separated into free acid and ' b o u n d ' fractions (the latter presumed to be the glucosyl ester; Milborrow (1970)), by acid/base partitioning of the homogenate with dichloromethane (Zabadal 1974). Radioactivity in free acid and ' b o u n d ' ABA fractions was measured by scintillation counting as above.
Results
ABA Uptake. In a typical experiment, the endogenous ABA concentration of healthy tobacco leaves was 24_+2 ng g 1 fresh weight (mean + standard error). Comparable, TMV-infected leaves contained 4 6 + 6 ng g-1. Endogenous ABA concentration varied with leaf age and environmental conditions: over all experiments the range was from 10-40ng g t in healthy leaves and 20-80 ng g a in TMV-infected leaves. Infected leaves consistently contained between two and five times the ABA concentration of comparable healthy leaves sampled at the same time. Leaf discs took up ABA from the culture medium for at least 24 h at a constant rate. After 24 h exposure to [I~C]ABA, 40% of the ABA taken up was recovered as the free acid and 60% was as the presumed glucosyl ester form, considered to be the major rapidstorage form of ABA (Milborrow 1970). This ratio of free to bound ABA was independent of exogenous ABA concentration. Uptake of ABA varied somewhat from experiment to experiment; overall it depended strongly on ABA concentration in the medium over a very wide range (Fig. 1). Discs from TMV-infected leaves consistently took up more ABA than comparable discs from healthy leaves: over all experiments ABA uptake by infected discs was 3.3_+ 0.3 times (mean + S.E.) that in healthy discs.
R.J. W h e n h a m and R.S.S. Fraser: A B A and R N A Synthesis
•'•10
e
10 ~
./
.-~,~104 103
./S ~
,~%~10=
