Planta (Berl.)105, 213--224 (1972) 9 by Springer-Verlag 1972

The Effects of Abscisic Acid on Senescence in Leaf Discs of Radish, Raphanus sativus L. A. J. Colquhoun a n d J. 1~. H i l l m a n Dept. of Physiology and Environmental Studies, University of Nottingham, Sutton Bonington, Loughborough, Leics., U.K. Received February 2, 1972

Summary. After a 6 day incubation period, abscisic acid (ABA) at 10-4 M retarded the decline in pigment levels and promoted the decline in protein levels of radish leaf discs. ABA treatment also retarded the rise in the specific activity of the RNA fraction (calculated by counts per minute incorporated of 14C-8-adenine as a fraction of optical density at 260 nm) observed in water-treated control discs. The results indicated that ABA was primarily effective in enhancing senescence in the early stages following leaf excision. Thus the increase in RNA specific activity during an initial 24 h incubation period was especially pronounced with ABA treatment although there was no effect of the hormone on RNA level. Moreover, in contrast to control discs, the pigment levels declined markedly in ABA-treated discs in this period. When the discs had been incubated in water ("preaged") for 3 or 5 days prior to ABA treatment, however, the hormone then had little effect on RNA metabolism and protein and pigment levels relative to the water control. Data are collated from different experiments to show the changes in RNA, pigment and protein with ABA treatment during a 6 day senescence period. I t is considered that ABA is speeding up the natural changes in RNA metabolism possibly by affecting both RNA synthesis and degradation. Introduction The most w i d e l y used a n d a c c e p t e d p a r a m e t e r s of leaf senescence are t h e decline in chlorophyll, p r o t e i n a n d R N A levels. I n these respects, t h e cytokinins, a u x i n s a n d gibberellins h a v e generally been f o u n d to be effective in r e t a r d i n g lea~ senescence (e.g. R i c h m o n d a n d Lang, 1957; Osborne a n d H a l l a w a y , 1959; F l e t c h e r a n d Osborne, 1965). More recently, i t has been r e p o r t e d t h a t abseisic acid (ABA) is effective in s t i m u l a t i n g chlorophyll loss in t h e leaves of a n u m b e r of p l a n t species (E1-Antably et al., 1967; Beevers, 1968). A B A has also been shown to enhance t h e p r o t e i n a n d R N A loss c h a r a c t e r i s t i c of senescence (Beevers, 1968 ; W a r e i n g et al., 1968). I t is n o t clear, however, w h e t h e r A B A t r e a t m e n t can a c t as a trigger for senescence or w h e t h e r i t affects t h e progress a n d m a i n t e n a n c e of senescence in t h e l a t e r stages. F u r t h e r , a l t h o u g h A B A is a p p a r e n t l y capable of s t i m u l a t i n g senescence in isolated leaf discs, little is k n o w n

214

A . J . Colquhoun and J. R. Hillman:

a b o u t f r a c t i o n s or c o m p o n e n t s of leaf m e t a b o l i s m w h i c h are a f f e c t e d b o t h in t h e p r i m a r y a n d l a t e r s t a g e s of t h e process. T h i s a r t i c l e r e - e x a m i n e s t h e effects of A B A t r e a t m e n t on leaf discs of radish, t h e s e n e s c e n c e of w h i c h h a s p r e v i o u s l y b e e n r e p o r t e d to be prom o t e d b y A B A t r e a t m e n t ( W a r e i n g et al., 1968; P a r a n j o t h y a n d W a r e i n g , 1971). I n t h i s article, h o w e v e r , c o n s i d e r a t i o n was m a d e of d e t a i l e d t i m e course s t u d i e s of c h a n g e s in c h l o r o p h y l l , p r o t e i n a n d R N A levels o v e r a p e r i o d of 6 d a y s as well as i n c o r p o r a t i o n of 14C-8-adenine i n t o t h e R N A fraction. M a t e r i a l s and Methods The plant material used was fully-expanded healthy leaf tissue from plants of

Raphanus sativus L. var. "Cherry Belle". The plants were grown in a glasshouse (temperature range 10-20 ~C) in natural light from April 1970 to October 1970 and with supplementary illumination from a bank of Phillips 400 watt mercury vapour lamps providing a 16 hr daily photoperiod from October 1970 to April 1971. All plant material was surfaee-sterilised in a 50 % solution of sodium hypoehlorite for 45 sec, followed by 6 rinses in distilled water. Discs 1 cm in diameter were punched from surfaee-sterilised leaves using a cork borer, taking care to avoid the main veins of the leaves. The discs were then placed in a beaker containing distilled water for a period not exceeding 30 min. Experiments involving incubation of discs for varying periods of time were carried out in 5 cm plastic petri dishes, each containing 10 randomly selected leaf discs and 10 ml of liquid, with the discs floating adaxial side uppermost. Incubation was carried out in a growth room at 25~ in darkness. RS-ABA was supplied by Hoffman La Roche, Basle, Switzerland. 14C-8-adenine (specific activity 51.1 fzCi/mM and 59.0 fzCi/mM) was obtained from the Radiochemical Centre, Amersham, Bucks. Typically, 13.2 mg of solid ABA was dissolved in 0.2 ml of methanol and made up to 500 ml with distilled water to provide a 10 ~ M solution of pH 4.2. This solution was stored at I~ in the dark until use, no solution being kept for longer than 4 weeks. The assay techniques for protein and RNA were modified by Beevers (pers. comm.) after Osborne (1962). Ten leaf discs were floated adaxial side uppermost in 5 ml of 14C-8-adenine solution in a 5 cm petri dish. The adenine solution contained 0.1 ~Ci of radioactivity in 0.01 ml of stock solution. The discs were gently infiltrated under vacuum for 1 rain and then incubated in a light room at 25~ for 4 h. After drying on filter paper, the discs were placed in tapered centrifuge tubes containing 5 mls of 80% ethanol and boiled for 6 min; this procedure was repeated twice, decanting the alcoholic solution each time. The discs were then homogenized in 5 ml ethanol using a hand-grinder. The homogenate was boiled for 6 min, centrifuged, and the supernatant decanted; this process was repeated twice. The pooled supernatants were made up to 50 ml with ethanol and the absorbance (O.D.) at 665 nm determined. The pellets remaining in the centrifuge tubes were washed twice each in 5 ml of ice-cold 20% trichloracetic acid, 5 ml ice-cold ethanol, and finally 5 ml of ether/ ethanol/chloroform (2:2:1) at room temperature. The replicates were then divided into two, half being used for a protein assay and the other half for RNA assay. For the protein assay, the pellet was suspended in 5 ml of 1 N NaOH solution and boiled for 10 rain in a water bath. After centrifugation of the extract, 1 ml aliquots of a colorimetrie solution (consisting of 100 2% sodium carbonate solution

Effects of Abscisic Acid on Senescence

215

in 0.1N NaOH:I 0.5% CuSO~ solution:l 1% sodium potassium tartrate) were added to test tubes to which 0.1 ml of the extract supernatant was added. After incubation at 35 ~C for 15 rain, 0.1 ml of 1 N Folin and Ciocalteus' reagent was added. After incubation in the dark at room temperature for 30 min, the colorimetric mixture was diluted to 10 ml with distilled water and the absorbance at 660 nm determined. For the RNA assay, the pellet was suspended in 5 ml of 0.3 M KOH solution and then incubated at 35 ~C overnight. The supernatant was collected and the pellet washed twice with 1 ml distilled water adding the washings to the original bulk supernatant. The supernatant was adjusted to pH 4.0 using 10% perchloric acid and the volume was made up to 10 ml with distilled water. After clarification of the solution by low speed ceutrifugation, the O.D. of the supernatant was determined at 260 nm and 1 ml aliquots were pipetted into scintillation bottles for subsequent radio-assay. Liquid scintillation assay for RNA was performed using a Nuclear-Chicago Unilux II liquid scintillation counter. The scintillation liquid consisted of toluene: Triton X-100 (2:1) with 4 gm of 2,5 diphenyl oxazole per litre of toluene; 8 ml of this mixture were added to each scintillation bottle. Each sample was counted for 2 periods each of 10 rain duration. Specific activity ratios were calculated as (cpm)/(O. D.).

Results Long Term Incubation Treatments Leaf discs were i n c u b a t e d for 6 days in either 10 ml of distilled w a t e r or 10-4M A B A . I n c o r p o r a t i o n of laC-8-adenine was t h e n determined. l~eference was m a d e to fresh leaf discs e x t r a c t e d in t h e same manner. Six replicates of t e n discs each were used an d t h e e x p e r i m e n t r e p e a t e d t h r ee times. D u r i n g 6 days i n c u b a t i o n t h e leaf discs e x h i b i t e d t h e chlorophyll loss characteristic of foliar senescence (Table 1). I n c o n t r a s t to previous evidence, h o w e v e r (E1-Antably et al., 1967 ; Beevers, 1968), it appears t h a t A B A has r e t a r d e d chlorophyll decline in comparison to w a t e r - t r e a t e d discs. F u r t h e r , after 6 days, it was seen t h a t A B A - t r e a t e d discs were limp b u t still m a r k e d l y green, whereas th e w a t e r - t r e a t e d discs were stiff

Table 1. The effect of ABA treatment on the optical densities of the chlorophyll (665 nm), protein (660 nm) and I~NA (260 nm) fractions, and on the incorporation of radioactivity into RNA Treatment

Chlorophyll (O.D. 665 nm)

Protein (O.D. 660 nm)

RNA (O.D. 260 nm)

RNA c.p.m,

I~NA, specific activity

Fresh

0.217 (:~0.005)

0.104 (•

1.19 (•

71.4 (•

60.0 ( i 5 . 3 )

6 day ABA

0.156 (~0.006)

0.050 (•

0.46 (•

6 days H20

0.115 (•

0.072 (--0.003) 0.50 (~0.08)

Data are shown with associated standard errors.

92.8 (• 159.1 (:~7.5)

2 0 1 .7 (• 318.2 (•

216

A.J. Colquhoun and J. R. Hil|man:

and still turgid, but had yellowed considerably. These data are also consistent with similar evidence obtained using Mackinney's method (1940) of chlorophyll extraction (Colquhoun and Hillman, unpublished.) Also from Table 1 it is evident that the protein level of the leaf discs has declined sharply during 6 days incubation especially in ABA-treated discs. Moreover, the RNA level of the discs declined markedly, and the specific activity of the RNA fraction was considerably increased by incubation relative to the value obtained for fresh discs. The specific activity of RNA from ABA-treated discs was markedly lower than from water-treated discs.

Preageing Discs in Water/or 3 Days Were ABA necessary to either initiate or regulate disc senescence, then it m a y be postulated that treatment of the leaf discs with this hormone at different stages of senescence might be expected to modify the process. Thus, the effects of ABA treatment were tested on leaf discs which had been pretreated ("preaged") in water for 3 days and which could be considered to be seneseing. Leaf discs were incubated in distilled water for 3 days and then transferred to 10 -4 M ABA for a further 3 days incubation period. Control discs were also transferred to fresh water at the same time. Extraction of the discs was carried out at 0, 3 and 6 days (Table 2). In agreement with Table 1, the optical density at 665 nm declined progressively from the level in fresh discs through 3 days to 6 days. I t is of note that the decline in optical density for preaged and control discs was similar at 6 days. Further, at 6 days there was little difference in protein levels between preaged and control discs. There was also an initial sharp decline in RNA level to 3 days in water-treated discs, with a subsequent slower decline to 6 days. The levels of t%NA in control and preaged discs at 6 days were similar. There was a sharp increase in specific activity with time; the activities were similar in 3 days and 6 day control discs and in preaged discs treated with ABA.

Preageing Discs in A BA Although ABA does not apparently affect the course of senescence in discs preaged in water (Table 2) it is feasible that ABA is not involved in the regulation of the senescence process once in progress but m a y act either as an initiating factor or be involved in the early stages. To investigate this possibility, comparison was made between discs treated in water and discs preaged in ABA and then transferred to water. Discs were thus first incubated for 3 days in 10 ml of 10 -4 M ABA and then transferred to distilled water for another 3 days prior to assay.

Effects of Abscisie Acid on Senescence

217

Table 2. The effect of preageing discs before ABA treatment on the optical densities of the chlorophyll, protein and RNA fractions, and on the incorporation of radioactivity into RNA Treatment

Chlorophyll (O.D. 665 nm)

Protein (O.D. 660 nm)

RIgA (O.D. 260 nm)

RI~A e.p.m,

RNA, speeific activity

0.234 (4-0.004) 0.129 (4-0.005) 0.76 (4-0.05) 3 days H~O 0.164 (4-0.009) 0.097 (4-0.008) 0.30 (4-0.13)

33.2 (4-2.6) 102.9(4-5.2)

43.7 (4-3.6) 343.0(•

6 days H~O 0.126 (4-0.008) 0.069 (4-0.003) 0.22 (4-0.05) 3 days H20 0.122 (4-0.006) 0.061 (4-0.004) 0.19 (4-0.03) 3 days ABA

85.9 (4-6.5)

390.5(4-46.4)

66.1 (4-5.6)

347.9 (4-45.7)

Fresh

Table 3. The effect of transferring ABA-treated discs to water on the optical densities of the chlorophyll, protein and RlqA fractions, and on the incorporation of radioactivity into RNA Treatment

Chlorophyll (O.D. 665 nm)

Protein (O.D. 660 nm)

RNA (O.D.260 nm)

Fresh

0.353 (4-0.012) 0.119 (4-0.011) 0.83 (•

RNA c.p.m, 25.6 (I1.4)

RNA, specifie activity 30.8 (12.0)

3 days ABA 0.256 (4-0.008) 0.068 (4-0.003) 0.35 (4-0.03) 3 days H~O 0.274 (4-0.006) 0.089 (4-0.006) 0.70 (:[:0.04) 3 days ABA 0.217 (4-0.011) 0.061 (4-0.003) 0.31 (4-0.04) 3 days H~O

33.8 (4-3.9)

96.6 (4-9.4)

73.9 (:t:4.8)

105.6 (4-4.2)

51.0 (4-0.2)

164.5 (4-17.1)

6 days H20 0.192 (4-0.006) 0.059 (4-0.003) 0.37 (4-0.04)

75.7 (•

204.6 (4-14.2)

The data in Table 3 show t h a t after 3 days ABA has apparently slightly stimulated the decline of the pigment fraction, relative to watertreated discs, i.e. apparently the reverse of the situation noted after 6 days incubation. After a further 3 days incubation, the pigment levels of discs preaged in ABA were similar to those of discs treated in water all the time. B y 3 days ABA had significantly increased the decline in the protein fraction (Table 3) but after a further 3 days, there was little difference between preaged and control discs in this respect. Table 3 also shows t h a t although at 3 days the level of R N A was much lower in ABA-treated discs than in control discs, the specific activities of this fraction from the 2 treatments were similar. The subsequent decline in R N A levels over the next 3 days of treatment was much slower in ABA-pretreated discs than in control discs up to 6 days when the levels of R N A were similar in the 2 treatments. The specific activity 15

Planta (Berl.), Bd. 105

218

A.J. Colquhoun and J. R. Hillman:

of the RNA fraction from ABA-treated discs was slightly lower than that from control discs.

Further Water-Preageing Treatments The results reported above suggest that ABA had little effect on the course of senescence in discs previously aged in water for 3 days. I t was considered that substantiation for these earlier observations could be obtained by using a preageing treatment intermediate between 3 days and 6 days. Thus discs were aged for 5 days in distilled water and then transferred for 1 day to ABA solution. ABA significantly retarded the decline in pigment level during a 5 day incubation period relative to control discs (Table 4). Subsequent ABA treatment for 1 day did not affect the level of this fraction. ABA also slightly increased the rate of protein decline during a 5 day incubation. Although the level of RNA decreased sharply with ageing, little difference was observed between the treatments at 5 and at 6 days (Table 4). Examination of the specific activities for RNA, however, show that the increase in activity previously observed with ageing is less marked in ABA-treated discs after 5 days than in water-treated discs. There is little difference in specific activity between the RNA fractions from 5 and 6 day water treatments and from preaged discs.

Short Preageing Periods in Water One day preageing treatments were used to study the effect of ABA in the very early stages of senescence. During this period, ABA substantially enhanced the rate of decline of the pigment fraction as compared with discs treated in water for the same time (Table 5). Subsequent treatment of water-aged discs with ABA for 5 days slowed the decline of this fraction in relation to control discs. After 1 day there was little decline in protein levels in either ABA- or water-treated discs. B y 6 days, the protein level had declined a little further in preaged discs than in control discs. There was also little change in RIgA level during a 1 day incubation period in either ABA or water (Table 5). There were marked increases, however, in the specific activity of the RNA, especially in the ABAtreated discs. B y 6 days, the level of RNA had declined to a simuilar value in both water-treated and preaged discs, although the specific activity was considerably lower in the latter.

Composite Graphs In Fig. 1, data from all experiments have been collated with regard to pigment, protein and RNA levels and presented as composite graphs

Effects of Abscisic Acid on Senescence

219

Table 4. The effect of preageing discs in water for 5 days before ABA treatment on the optical densities of the chlorophyll, protein and l~qA fractions, and on the incorporation of radioactivity into t~NA Treatment

Chlorophyll (O.D. 665 nm)

:Protein (O.D. 660 rim)

RNA (O.D. 260 nm)

Fresh

0.323 (4-0.006) 0.118 (•

5days 1t20

0.113'(4-0.007) 0.075 (4-0.003) 0.33 (4-0.02)

1.01 (4-0.08)

t~NA c.p.m, 29.9 (•

P~NA, specific activity 29.6 (4-1.3)

104.1 (4-6.7)

315.3 (4-32.4)

0.064 (4-0.004) 0.31 (4- 0.04)

45.4 (4- 2.9)

146.5 (4- 6.4)

6 days H,O

0.095 (4-0.010) 0.076 (4-0.003) 0.31 (4-0.01)

101.0 (4- 3.7)

325.8 (4-33.6)

5 days ~ 0 1 day ABA

0.108 (4-0.008) 0.067 (•

101.2 (4-5.5)

337.3 (4-41.2)

5 days ABA 0.195 (4-0.016)

0.30 (•

Table 5. The effect of preageing discs in water for 1 day before ABA treatment on the optical densities of the chlorophyll, protein and I ~ A fractions, and on the incorporation of radioactivity into RBTA Treatment

Chlorophyll (O.D. 665 nm)

Fresh

0.430 (4-0.028) 0.131 (4-0.008) 0.57 (4-0.02)

1 day H20

0.352 (4-0.009) 0.159 (4-0.005) 0.58 (4-0.03)

83.9 (4-7.8)

144.7 (4-9.4)

1 day ABA

0.287 (4-0.011) 0.140 (4-0.004) 0.59 (4-0.05)

169.0 (4-10.7)

286.4 (4-22.6)

6 days H20

0.084 (4-0.007) 0.076 (4-0.003) 0.33 (4-0.02)

138.0 (4-8.3)

418.3 (4-22.2)

66.8 ( • 6.5)

196.5 (4-15.6)

1 day I-I20 0.163 (• 5 days ABA

Protein (O.D. 660 nm)

RNA (O.D. 260 nm)

0.063 (4- 0.004) 0.34 ( • 0.01)

I~NA c.p.m, 19.8 (4-4-0.8)

I~NA, specifie activity 34.8 (4-8.6)

in which absorbance values have been expressed relative to fresh discs from the same experiment. Fig. 1 A shows that between 0 and 3 days, the level of pigments is significantly higher in water-treated discs than those aged in ABA. At about 3 days, however, a reversal of this situation occurs until at 5 and 6 days the level of pigment is significantly higher in ABA treatments than in control treatments. Protein levels decline sharply with age, this decline being apparently faster in ABA-treated discs (Fig. 1B). The consequent differential in protein levels between the two treatments appears to be set up during the first 24 h of incubation. There is no difference between ABA- and water-treated discs with respect to RNA levels either at 1 day or later at 5 and 6 days (Fig. 1 C). 15"

220

A.J. Colquhoun and J. R. Hillman: 1.2-

A

/ ~ \

S

C

~: 0.8r,,,"

d 0.6d 0.40.20

I 0

1

[ 2

1

I 4

I

[ 6

] 0

1 TIME

1 2

1

1 4

IN

I

[ 6

I

I

I 2

I

I 4

I 6

DAYS

Fig. 1. A--C. Composite graphs of- A pigment levels (O. D. 665 nm); B protein levels (O.D. 660 nm); C RNA levels (O.D. 260 am), of radish leaf discs during a 6 day incubation period. Absorbance values expressed as a ratio of levels in flesh discs where fresh levels equivalent to 1.00. 9 9 water control; o oABA treatment. Vertical bars equal to twice standard error.

After 3 days, however, RNA levels are slightly lower in ABA-treated discs than in water-treated discs. Discussion The observation that ABA apparently retarded chlorophyll decline in relation to control discs after 6 days incubation (Table 1) appears to contradict the findings of E1-Antably et al. (1967) and Beevers (1968). However, analysis of Fig. 1 A shows that the position is complex and that in the early stages of incubation ABA appears to enhance the rate of chlorophyll decline; thus for short incubation periods, our data do agree with the published data. The reversal in ABA effect occurring after about 3 days, on the other hand, is unsubstantiated in the literature. The observation that ABA-treated discs were limp and delicate after 6 days incubation, whereas water-treated discs were still stiff and turgid, may relate to the hormone affecting cell membranes and possibly permeability (Eflam, 1965; Glinka and Reinhold, 1971). In general agreement with the published data, protein levels were shown to decline in leaf discs with ageing (e.g. Osborne, 1962) and ABA was found to speed up the decrease in protein levels (Beevers, 1968). During 6 days incubation, RNA levels decreased markedly in watertreated and ABA-treated discs and in discs subjected to the various "preageing" treatments. The decrease in RNA levels was slightly faster in ABA treatment after 3 days. Table 1 shows that both incorporation into and specific activity of the RNA fraction increased with ageing, the

Effects of AbscisicAcid on Senescence

221

increases in both parameters being less after 6 days in ABA-treated than in water-treated discs. ABA has little apparent effect on the senescence process advanced experimentally by "pre-ageing" (Table 2). Thus, after 3 and 5 days preageing in water, ABA did not alter the decline of RNA, pigment or protein levels or the increase in specific activity of the RNA fraction. These data suggest that ABA may be involved in the primary stages of senescence. From i day incubation experiments (Table 5) an early effect of ABA in disc senescence is to increase markedly the rate of turnover of RNA in relation to both fresh and water-treated discs without affecting the RNA level. Other data (Table 3) show that ABA appears to slightly stimulate RNA decline after 3 days relative to water-treated discs. Further, by 6 days the specific activity of RNA in control discs increases beyond the value obtained at 1 day, whereas in preaged discs, treated in days 1 to 6 with ABA, the specific activity decreases again. Thus, ABA has an effect on the specific activity of the RNA fraction at an early stage of senescence. Since at 5 and at 6 days the specific activity of the RNA is lower when treated in ABA, it therefore appears that ABA is acting to speed up the process of RNA decline. ABA treatment, then, appears to cause an initial increase in RNA turnover which is subsequently reduced, whereas water treatment leads to a slower but steadier increase in specific activity during the 6 day treatment period. There is disagreement in the literature with regard to the changes in the levels of RNA and protein during the early stages of senescence (i. e. during the first 24 h after excision). Sacher (1965) reported that in excised bean pod tissue, the decline in RNA proceeded from zero time and that by 15 h 20% of the RNA had disappeared. In contrast, Srivastava and Ware (1965), using excised barley leaves, noted an initial increase in RNA levels followed by a subsequent decline after about 2 days. In our experiments there was little change in RNA levels during 1 day incubation and protein levels increased only slightly. In agreement with previous reports the specific activity of RNA increased with ageing relative to the specific activity of fresh discs (Cherry etal., 1965; Srivastava and Atkin, 1968), with ABA stimulating this increase at short incubation periods (1 day) but retarding it with longer incubation times. Beevers (1968) found that ABA did not alter the incorporation of 14C-adenine into RNA with ageing, but noted an increase in specific activity of RNA as the level declined. On the other hand, Wareing et al. (1968) found that ABA inhibited 3~p incorporation into all fractions of RNA with a specific effect on heavy ribosomal RNA. However, the qualitative effect of ABA on RNA precursor incorporation may depend

222

A.J. Colquhoun and J. R. Hillman:

on experimental conditions and plant tissue (Pietro and Sacher, 1970) as well as length of treatment. Although we appreciate the possible effect of bacterial contamination on our incopporation data (Burdett and Wareing, 1968), we see little reason on this basis for doubting the validity of the differences observed between preaged, ABA- and water-treated leaf discs. These conclusions are generally substantiated by unpublished data obtained using an antibiotic mixture consisting of streptomycin, mycostatin and penieilhn to control contamination. Since increases in adenine incorporation and RNA specific activity, and a decline in RNA levels occur during senescence of the discs, it is concluded that both RNA synthesis and degradation are increased during incubation. Pietro and Sacher (1970) have recently demonstrated that ABA increased both RNA-ase and acid phosphatase activities in senescing Rhoeo leaf sections, with most of the rise in RNA-ase level occurring during the first 6 h after excision. Despite this, they consider it unlikely that the only role of ABA in accelerating senescence is due to an effect on RNA-ase but suggest that the inhibition of synthesis of total RNA and protein by ABA may contribute to senescence. Our data, however, show that an increase in synthesis of RNA occurs during senescence; this is substantiated indirectly by the increases in hydrolytic enzymes observed by e.g. Srivastava and Ware (1965), Lewing~on et al. (1967). Thus, ABA may selectively affect specific RNA species and proteins. Trewavas (1968) suggested that the level of ribosomes or r-RNA in seneseing leaf tissue control the rate of protein synthesis and that hormones might increase or decrease the rate of protein synthesis by regulating the production of r-RNA. ~urther, Trewavas (1970) has shown that in Lemna minor, ABA reduced the rate of r-RNA synthesis with little or no effect on the rate of degradation. Paranjothy and Wareing (1971) found, however, that senescence in radish leaves was not altered by the inhibition of r-RNA synthesis with 5-fluorouraefl. They therefore consider that there is little prospect of the decline in protein being attributable to any inhibitory action of ABA, and suggest that the effects of ABA on protein synthesis are largely due to regulation of polydisperse and s-RNA synthesis. Nevertheless, in earlier work, Wareing et al. (1968) showed that ABA reduced the level of polysomes in leaf discs without affecting the level of monomeric ribosomes. I t is thought that about 75-80% of the RNA of plant cells consists of r-RNA (see Clowes and Juniper, 1968). Thus, if the 1/~-life of r-RNA is considerably longer than that of other RNA fractions (Trewavas, 1970) and since RNA levels have been observed to decline to about 40 % of original levels in our experiments, then our data may indicate that ABA

Effects of Abseisic Acid on Senescence

223

could be having a greater effect on the synthesis of r-RNA than on its degradation. We thank Professor M. B. Wilkins for his interest and encouragement, and Mr. J. Blunt and Mr. ft. Topham for expert technical assistance.

Reterenees Beevers, L. : Growth regulator control of leaf senescence in leaf discs of nasturtium (Tropaeolum ma]us). In: Biochemistry and physiology of plant growth substances, p. 1417-1435 (F. Wightman and G. Setterfield, eds.). Ottawa: Runge Press 1968. Burdett, A. N., Wareing, P. F. : The effects of kinetin and contaminating bacteria on the incorporation of ~ into various fractions of nucleic acids extracted from radish leaves. Planta (Berl.) 81, 88-96 (1968). Cherry, J . H . , Chroboezek, H., Carpenter, W. J. G., Richmond, A.: Nucleic acid metabolism in peanut cotyledons. Plant Physiol. 40, 582-587 (1965). Clowes, F. A. L., Juniper, B. E.: Plant Cells, p. 112. Oxford: Blackwell Scientific Publications 1968. Eilam, Y. : Permeability changes in senescing tissue. J. exp. Bot. 16, 614-627 (1965). E1-Antably, It. M. M., Wareing, P. F., Hillman, J. : Some physiological responses to d,l abscisin. Planta (Berl.) 78, 74-90 (1967). Fletcher, 1~. A., Osborne, D. J. : Regulation of protein and nucleic acid synthesis by GA during leaf senescence. Nature (Lond.) 207, 1176-1177 (1965). Glinka, Z., Reinhold, L.: Abscisic acid raises the permeability of plant cells to water. Plant Physiol. 48, 103-105 (1971). Lewingten, R. J., Talbot, M., Simon, E. W.: The yellowing of attached and detached cucumber cotyledons. J. exp. Bot. 18, 526-534 (1967). Maekinney, G. : J. biol. Chem. 132, 91 (1940). Cited by Paech and Tracey in: Modern methods of plant analysis, IV, p. 143. Berlin-G6ttingen-Heidelberg: Springer 1955. Osborne, D . J . : The effect of kinetin on protein and nucleic acid metabolism in Xanthium leaves during senescence. Plant Physiol. 37, 595-602 (1962). Osborne, D. J., Hallaway, tt. M. : Role of auxins in the control of leaf senescence. In: 4th International conference of plant growth regulators, p. 329-340 (W. Klein, ed.). New York: Iowa State Press 1961. Paranjothy, K., Wareing, P. F. : The effects of ABA, kinetin and 5-fluorouraeil on RNA and protein synthesis in senescing radish leaf discs. Planta (Berl.) 99, 112-119 (1971). Pietro, D. L., Saeher, J. A. : Control of ribonuclease and phosphatase by auxin and ABA during senescence of Rhoeo leaf sections. Plant Physiol. 46, 806-811 (1970). Richmond, A. E., Lang, A. : The effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125, 650-651 (1957). Sacher, J . A . : Senescence: hormonal control of RNA and protein synthesis in excised bean pod tissue. Amer. J. Bot. 52, 841-848 (1965). Srivastava, B. I. S., Ware, G. : The effect of kinetin on nucleic acids and nueleases of excised barley leaves. Plant Physiol. 40, 62-64 (1965). Srivastava, B. I. S., Atkin, R. K. : Effect of second leaf removal or kinetin treatment on the nucleic acid metabolism of the sencseing first seedling leaf of barley. Biochem. J. 107, 361-366 (1968).

224

A . J . Colquhoun and J. R. Hillman: Effects of Abscisic Acid on Senescence

Trewavas, A. : Relationship between plant growth hormones and nucleic acid metabolism. Progr. Phytochem. 1, 113-160 (1968). Trewavas, A. : The turnover of nucleic acids in JLemna minor. Plant Physiol. 45, 742-751 (1970). Wareing, P. F., Good, J., Potter, H., Pearson, J.A.: Preliminary studies on the mode of action of ABA. In: Plant Growth Regulators, p. 191-205. 31: Society of Chemical Industry Monograph 1968. A. J. Colquhoun J. R. Hillman Department of Botany University of Glasgow Glasgow, W.2., U.K. (present address)