Planta (Berl.) 92, 178--188 (1970)

Changes in Endogenous Growth Regulators in Nasturtium Leaves during Senescence TINo-YuN CHIN and LEONARD BEEVERS Department of Horticulture, University of Illinois at Urbana-Champaign, Urbana Received March 6, 1970

Summary. By use of lettuce-hypocotyl and wheat-coleoptile bioassay, the presence of both gibberellin (GA)-like and abseisic-acid(ABA)-like components in acidic ethyl-acetate extracts of fully expanded nasturtium (Tropaeolum ma]us) leaves has been shown. During senescence of detached leaves there was a progressive decline in GA-like components and an increase in ABA-like components. Pretreatment of detached leaves with GAa or kinetin prevented changes in the levels of endogenous growth regulators and delayed senescence. The observations provide experimental verification for the concept that senescence is associated with changes in endogenous growth regulators. Introduction

During leaf senescence chlorophyll content declines and the leaves develop characteristic colorings. These changes occur at an accelerated rate in detached leaves. However, the onset of senescence symptoms can be arrested by the application of various regulatory compounds. Thus, following the original observation of Richmond and Lang (1957) that kinetin treatment retarded the senescence of detached leaves of X a n t h i u m it has been demonstrated that kinetin will delay the senescence of detached leaves of a wide range of species (for example, Osborne, 1962; Sugiura et al., 1962; Wollgiehn, 1961). I n addition, auxin (Osborne and Itallaway, 1960) and gibberellins (GAs) (Fletcher and Osborne, 1965; W h y t e and Luckwill, 1966; Beevers, 1966) have been reported to retard senescence of leaves of certain species. I n contrast, abseisic acid (ABA) has been shown to accelerate senescence of detached leaves (E1-Antably, et al., 1967; Beevers, 1968). These observations have led to the development of the concept that senescence rate is controlled by the level of endogenous growth regulators. Thus a decline in the endogenous level of auxin, GAs or cytokinins, or an increase in ABA would accelerate senescence. In detached leaves senescence would be accelerated due to the depletion of endogenous senescence retarding growth regulators. However there has been no experimental confirmation of this idea although the findings of Fletcher et al. (1969) that endogenous gibberellie acid (GAa) levels decline with leaf age in dandelions (Taraxacum o]]icinale)

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support the proposition. The present investigation attempts to study the changes in endogenous growth regulators during leaf senescence and to relate senescence rate to growth regulator content.

Materials and Methods Plant Material; Extraction; Chromatography. Nasturtium ( Tropaeolum ma]us L.) leaves were used for the extraction of growth regulators. Leaves (approximately 6 cm in diameter) were collected at intervals as required. Some leaves were extracted immediately after harvest and t a k e n as zero-day controls; other leaves were placed in plexiglass containers (13 • i0 • 4 = ca. 33 • 26 X .5.5 cm) lined with wet paper toweling, a n d kept in darkness at 25 ~. The leaves were sprayed with water from a n atomizer every 2 days in order to prevent wilting. Samples were removed for the extraction of growth regulators at 2, 4 a n d 8 days. I n studies involving GAa or kinetin t r e a t m e n t of the leaves, GA 3 or kinetin (20 mg/l in 0.005% Tween-20) were sprayed on the lower leaf surface a t a rate of 2 ml per leaf immediately after collection. This t r e a t m e n t was repeated after 3 days. Leaves treated with 0.005% Tween-20 solution were t a k e n as controls. All the treated leaves were incubated in moistened Plexiglass containers and maintained for 8 days in the dark a t 25 ~ Prior to extraction, leaves were frozen in liquid nitrogen and lyophilized. Subsequently growth regulators were extracted from 25 gm of dried material as described b y Zeevaart (1969). For paper chromatography of the extracts, a measured quantity of sample was streaked onto W h a t m a n No. 1 m m chromatography paper a n d the chromatogram was developed in the descending manner with a solvent of isopropanol/ammonia/ water (10:1 : 1 v/v) at room temperature. The solvent front was allowed to r u n 15 cm from the starting line. The paper was air dried a t room temperature a n d divided transversely into l0 equal strips. An additional strip below the starting line was used as a control. A volume of 0.5 ml of glass-distilled water was added to each strip a n d the paper was allowed to stand for several hours before bioassay. For t h i n layer chromatography (TLC), a measured volume of extract was strip-loaded onto 20 • 20 cm TLC plates of silica gel F-254 (E. Merck type precoated preparative glass plates with florescent indicator, layer of 0.5 mm, obtained from B r i n k m a n n Co., N.Y.). The plate was developed to 15 cm in a mixture of benzene/ethyl acetate/acetic acid (70:30:5, v/v) (Rudllicki, ]969). Ten equal zones of silica gel were scraped from the plate a n d eluted 3 times with water-saturated ethyl acetate. The eluates were evaporated to dryness a n d each sample was then redissolved in a small volume of distilled water. Bioassay. The distribution of GA-like activity in the chromatograms of leaf extracts was determined using the lettuce-hypocotyl assay (Frankland and Wareing, 1961). Ten lettuce seedlings (Lactuca sativa L., cv. Grand Rapids) were directly incubated on the chromatogram paper a n d the mean hypocotyl length was recorded 4 days after incubation. ABA-like activity was detected b y a modified wheat-coleoptile straight-growth assay (Bentley, 1954). Subapical coleoptile sections, 10 m m in length, were cut from wheat seedlings (Triticum aestivum L. cv. Victor) grown in the dark for 60 hr. ~ e n sections were placed on a filter paper containing 0.8 ml of the prepared test solutions from TLC plates, or known concentration of synthetic ABA. The coleoptile sections were kept in darkness at 25 ~ for 24 hr. The final length of the sections was measured b y means of a n overhead projector. Nasturtium leaf discs were used to examine the effect of the chromatographed leaf extracts on senescence. Leaf discs, 7 m m in diameter, were cut from fully

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expanded leaves with a cork borer. Ten discs were placed on each strip of the paper chromatogram moistened with 0.8 ml of distilled water in a Petri dish. The Petri dish was kept in a plexiglass container lined with moist filter papers, and incubated at 25~ in the dark. After 4 days of incubation the leaf discs were extracted with 80 % ethanol and the chlorophyll content of the extracts was determined spectrophotometrically (Beevers, 1966).

Results

1. Growth Regulators in _Nasturtium Lea/Extracts. Using the lettucehypocotyl bioassay to detect the distribution of growth regulatory compounds present in the acidic ethyl acetate fraction following separation by paper chromatography, it was found (Fig. 1A) that in extracts from freshly harvested leaves there was a significant component at Rf 0.4 0.6 on the chromatogram which stimulated hypocoty] growth. This zone corresponds to the location of authentic GA a chromatographed in this system. In addition to the growth-stimulatory material there was a region of the chromatogram at Rf 0.8--1.0 which showed slight growth inhibition. This zone corresponds to that of authentic ABA. The growth inhibitory compound was characterized more extensively when the acidic ethyl-acetate fraction was separated by TLC and used in the wheat coleoptile assay. The chromatogram zone at Rf 0.1--0.3 was inhibitory to coleoptile growth (Fig. 2 A). Authentic ABA was located at Rf 0.3 when chromatographed under similar condition. On the basis of these separations it is considered that at least some of the growth regulatory zone at Rf 0.4---0.6 on the paper chromatograms corresponds to GA, possibly GAs, and we will refer to this region as GAlike activity. The growth inhibitory zone Rf 0.8--1.0 on the paper chromatogram and Rf 0.1--0.3 on the thin layer chromatograms corresponds in part to ABA and these zones will be termed ABA-likc. 2. Changes in Growth Regulator Content during Senescence. Under the conditions of incubation used in the present experiment it was found that leaves became visibly yellow after 4 days and were completely yellow after 8 days. Thus, analysis of leaves 0, 2, 4 and 8 days after harvest should provide information on the growth-regulator content at strategic stages of the senescence process. I t was found that as leaves aged there were significant chages in the amounts of growth regulators extractable in the acidic ethyl acetate fraction. The lettuee-hypoeotyl assay (Fig. 1 A - - D ) indicated that there was only a trace of GA-like activity remaining by day 4, and no GA-likc activity at all was detected in the extract prepared from leaves incubated for 8 days. Associated with the decrease in growth-stimulatory components at Rf 0.4--0.6 on the chromatograms there was an increase in ABA-likc growth-inhibitory components at Rf 0.8--1.0. This was confirmed in the wheat coleoptile bioassay of material separated by TLC (Fig. 2 A ~ D ) .

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Fig. 1. Lettuce hypocotyl bioassay of gibberellin-likeactivity in acidic ethyl-acetate extracts of nasturtium leaves following separation by paper chromatography using isopropanol/ammonia/water (10:1 : 1 v/v). Extracts were prepared from leaves after 0, 2, 4 and 8 days incubation in the dark at 25~ Extract equivalent to 30 g of fresh weight was applied to each chromatogram. Broken lines irtdicate the 95 % confidence irtterval of the control

3. Changes in Endogenous Growth Regulators in Response to Exogenous Gibberellic Acid or Kinetin. Previous observation (Beevers, 1968) have shown t h a t GA s or k i n e t i n t r e a t m e n t can delay the senescence of n a s t u r t i u m leaf discs. I t was of interest, therefore, to s t u d y the influence of exogenous growth regut&tors on the endogenous GA- a n d ABA-like components. Leaves were t r e a t e d with k i n e t i n or GA 3 i n Tween-20 solution as described. E x t r a c t s were prepared from these leaves 8 days after harvest. A t this time the leaves which had been t r e a t e d with GA s were still green; those t r e a t e d with k i n e t i n showed some yellowing of the leaf blade, a n d the control leaves which h a d been sprayed with only Tween-20 were completely yellow.

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Fig. 4. ABA-like activity in chromatograms of acidic ethyl-acetate extracts from nasturtium leaves. Extracts were prepared from GAa-treated, kinetin-treated or control leaves after 8 days of incubation in the dark and separated by thin-layer chromatography on silica-gel-F plates using benzene/ethyl acetate/acetic acid (70:30:5 v/v) as solvent. Broken lines indicate the 95 % confidence of the control The lettuce-hypocotyl assay indicated that extracts from leaves which had been treated with GA 3 had a high content of GA-like material. (Fig. 3). Some of this material may represent the exogenously supphed GA 3 and thus it is difficult to determine whether there had been major changes in the endogenous GA level during the 8-day incubation period. I n the GA3-treated leaves there was no aecumulation of materials which were inhibitory to lettuce hypoeotyl elongation. Extracts from leaves which had been treated with kinetin had an appreciable content of GAlike material and a low content of ABA-like material. In contrast, extracts from the control leaves had no detectable GA-like components and had accumulated ABA-like components. I n assays for ABA-like components using the wheat-coleoptile test it was found that there was an appreciable level of this factor in extracts from control leaves (Fig. 4). However, the level of ABA-like components in extracts prepared from GA 3 or kinetin treated leaves after 8 days was similar to that of freshly harvested leaves.

4. EHect o[ Leaf Extracts on Senescence o/ Nasturtium Lea/ Discs. Extracts were prepared from control leaves incubated for 0, 2, 4 and 8 days, or from similar leaves treated with GA 3 or kinetin. The extracts were chromatographed and their influence on senescence determined. I t was found that the extracts prepared from freshly harvested leaves contained material which chromatographed at I~f 0.3--0.5 and which prevented the loss of chlorophyll from nasturtium leaf discs. The level of this component decreased as the leaves seneseed. Associated with the decrease in this component there was an accumulation of materials, ehromatogram zone at I~f 0.7--1.0 which accelerated senescence of the leaf discs (Fig. 5A).

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Fig. 5. A. The influence of chromatographed components of acidic ethyl acetate extracts of nasturtium leaves on retention of chlorophyll in senescing nasturtium leaf discs. Extracts were prepared from leaves following 0, 2, 4 and 8 days incubation in the dark and separated by paper chromatography using isopropanol/ammonia/ water (10:1:1, v/v). Nasturtium leaf discs were incubated in Petri dishes in the presence of extracts from chromatographic zones for 4 days and then chlorophyll content of 80% ethanol extracts of the leaf discs was determined I n c h r o m a t o g r a m s p r e p a r e d from e x t r a c t s of G A a - t r e a t e d leaves t h e r e was a region a t R f 0 . 3 - - 0 . 6 which effectively d e l a y e d t h e c h l o r o p h y l l loss f r o m n a s t u r t i u m leaf discs a n d a zone a t R f 0 . 7 - - 0 . 9 which o n l y slightly a c c e l e r a t e d senescence. I n e x t r a c t s p r e p a r e d f r o m k i n e t i n - t r e a t e d leaves t h e r e was also m a t e r i a l , c h r o m a t o g r a p h i n g a t R f 0.3--0.6, which r e t a r d e d senescence. H o w e v e r , this zone was n o t as effective as t h a t f r o m c h r o m a t o g r a m s of e x t r a c t s f r o m GA~-treated leaves, suggesting t h a t t h e a c t i v e principle was p r e s e n t a t a lower c o n c e n t r a t i o n . I n t h e e x t r a c t s from t h e k i n e t i n - t r e a t e d leaves t h e r e was also evidence of m a t e r i a l which c h r o m a t o g r a p h e d a t R f 0 . 7 - - 1 . 0 which s l i g h t l y a c c e l e r a t e d c h l o r o p h y l l loss from n a s t u r t i u m leaf discs. I n e h r o m a t o g r a m s of e x t r a c t p r e p a r e d f r o m control leaves 8 d a y s a f t e r h a r v e s t t h e r e was no evidence of m a t e r i a l which could d e l a y t h e

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chlorophyll loss from n a s t u r t i u m leaf discs. H o w e v e r , t h e r e was a comp o n e n t a t Rf 0 . 7 - - 1 . 0 which a c c e l e r a t e d senescence. This c o m p o n e n t a p p e a r e d to be p r e s e n t in m u c h greater q u a n t i t y in e x t r a c t s from t h e control leaves t h a n in e x t r a c t s from G A 3- or k i n e t i n - t r e a t e d leaves (Fig. 5B).

5. Presence o/ Growth Regulators in Naturally Seneseing Leaves. E x t r a c t s p r e p a r e d from yellow leaves which h a d senesced while still a t t a c h e d to t h e p l a n t c o n t a i n e d no GA-like a c t i v i t y ; on t h e o t h e r h a n d , t h e y d i d c o n t a i n A B A - l i k e m a t e r i a l which r e s t r i c t e d t h e g r o w t h of w h e a t coleoptiles a n d a c c e l e r a t e d t h e senescence of n a s t u r t i u m leaf discs (Fig. 6). Discussion The c h r o m a t o g r a p h i c analysis a n d ] e t t u c e - h y p o c o t y ] b i o a s s a y of t h e acidic e t h y l - a c e t a t e e x t r a c t s of n a s t u r t i u m leaves i n d i c a t e t h e presence of gibberellin-like compounds. This finding is consistent w i t h t h e r e p o r t s of L a n g (1960), I-Iarada a n d N i t s c h (1959), "Wheeler (1960), Z e e v a a r t (1969), a n d F l e t c h e r et al. (1969) of t h e occurrence of GA-like m a t e r i a l s in e x t r a c t s from leaves of o t h e r species. P a r t of t h e GA-like c o m p o u n d c h r o m a t o g r a p h e d a t t h e s a m e location as a u t h e n t i c GAa, suggesting t h a t a t least some of t h e GA-like a c t i v i t y m a y be due to GA a. F l e t c h e r et al. (1969) suggested t h a t t h e p r i n c i p a l G A in e x t r a c t s from Taraxacumo]/icinale leaves is G A 3 .

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Fig. 6A--C. Oibberellin- and ABA-like components separated by paper chromatography from acidic ethyl-acetate extracts of nasturtium leaves which had senesced while attached to the plant. A lettuce hypoeotyl assay; B wheat eoleoptile assay; C nasturtium leaf disc assay. Broken lines indicate the 95 % confidence interval of the control I n addition to the GAs, there is present in the acidic ethyl-acetate extracts a compound which restricts lettuee-hypocotyl elongation and growth of wheat-coleoptile segments. This material shows chromatographic properties similar to ABA. Thus, it appears that ABA-like components, although at low levels, are present in fully expanded nasturtium leaves. The occurrence of ABA-like components in leaves of other species has also been reported (Milborrow, 1967; Garr and Guttridge, 1968; Pieniazek and Rudnicki, 1967; Rudnicki etal., 1968; Wright, 1969). The material located on the paper chromatograms at Rf 0.3--0.6 efficiently retarded the senescence of nasturtium leaf discs whilst the material from g f 0.7--1.0 accelerated senescence in this tissue (Fig. 5B).

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Since these are t h e zones which show GA- a n d A B A - l i k e a c t i v i t y , respectively, in g r o w t h bioassays i t a p p e a r s t h a t t h e senescence of n a s t u r t i u m leaves could be controlled b y these endogenous g r o w t h regulators. D u r i n g t h e senescence of d e t a c h e d n a s t u r t i u m leaves, t h e GA-like c o m p o n e n t s decline while t h e r e is a n increase in A B A - l i k e constituents. Changes in these c o m p o n e n t s were e v i d e n t after i n c u b a t i n g t h e d e t a c h e d leaves for 2 d a y s ; these changes therefore p r e c e d e d t h e visible onset of senescence a n d yellowing which occurred a t t h e f o u r t h d a y of i n c u b a t i o n . G A a- or k i n e t i n - t r e a t m e n t s d e l a y e d senescence, p r e v e n t e d t h e increase in A B A - l i k e c o m p o n e n t s a n d m a i n t a i n e d t h e level of GA-like compounds. F l e t c h e r et al. (1969), on t h e basis of studies of changes in t h e endogenous G A c o n t e n t of a t t a c h e d d a n d e l i o n leaves, suggest t h a t senescence of these leaves is associated w i t h a deficiency of GAs. The o b s e r v a t i o n s t h a t G A c o n t e n t declines d u r i n g t h e senescence of n a s t u r t i u m leaves agree with this concept. H o w e v e r , i t is e q u a l l y a p p a r e n t t h a t senescence is a s s o c i a t e d w i t h an increase in A B A level. I n view of t h e o b s e r v e d a n t a g o n i s m of A B A a n d GAa (see A d d i c o t t a n d L y o n , 1969), it a p p e a r s t h a t t h e a c c u m u l a t e d A B A would become progressively m o r e effective in h a s t e n i n g senescence as t h e G A c o n t e n t declined in t h e leaf. An increase in ABA level in detached wheat leaves has recently been reported by Wright (1969) and Wright and ]-Iiron (1969). They have indicated that the accumulation of inhibitor could be associated with the wilting process. It is possible that the accumulation of ABA-like materials in the senescing nasturtium leaves could be partially associated with wilting. However, this possibility seems highly unlikely since the leaves were maintained in a closed atmosphere saturated with water vapor. Furthermore, the observation that ABA-like materials did not accumulate in uon-senescing leaves maintained under similar conditions suggests that the increase in inhibitor level is a function of senescence per se. The findings t h a t leaves which senesce while a t t a c h e d to t h e p l a n t h a v e no d e t e c t a b l e GAs a n d a c c u m u l a t e A B A - l i k e m a t e r i a l s suggest t h a t t h e changes in t h e levels of gibberellin a n d A B A - l i k e c o m p o n e n t s which occur in d e t a c h e d leaves a r e similar to those which occur in a t t a c h e d leaves a n d are n o t a n e x p e r i m e n t a l artifact. Such o b s e r v e d changes p r o v i d e e x p e r i m e n t a l verification for t h e concept t h a t leaf senescence is associated w i t h changes in levels of endogenous g r o w t h regulators. References Addicott, F. T., Lyon, J. L.: Physiology of abscisic acid and related substances. Ann. Rev. Plant Physiol. 20, 139--164 (1969). Beevers, L. : Effect of gibberellic acid on the senescence of leaf discs of nasturtium (Tropaeolum majus). Plant Physiol. 41, I074--I076 (1966). - - Growth regulator control of senescence in leaf disc of nasturtium (Tropaeolum ma]us). In: Biochemistry and physiology of plant growth substances, p. 1417-1436, F. Wightman and G. Setterfield, eds. Ottawa: Runge Press 1968.

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Chin and Beevers: Growth Regulators during Leaf Senescence

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Dr. Leonard Beevers Department of Horticulture University of Illinois Urbana, Illinois 61801, U.S.A.