Planta (Berl.) 102, 1-10 (1972) 9 by Springer-Verlag 1972

Uptake and Metabolism of Radioactive Gibberellins by Barley Aleurone Layers* ALAN MUSOI~AVV,, SANI)RA E. KAYs, and HA~S Kv,~D~ 1KSU/AEC Plant Research Laboratory, Michigan State University, East Lansing Received August 20, 1971 Summary. When aleurone layers were treated with labeled gibberellin A1 (3H-GA1), gibberellin A5 (3H-GA5) and the methyl ester of 3H-GA5 (3H-GAs-1VIE), radioactivity was accumulated by the tissue for a period of 20-30 h. After this time, radioactivity was released into the medium. Concomitantly, ribonuclease was also liberated by the tissue. The radioactivity accumulated by aleurone layers was associated with polar metabolites of the respective GAs, and the extent of accumulation was a function of the degree of GA metabolism (GAs-M_E> GAs> GA1). Accumulation of radioactivity was inhibited in the cold and by the metabolic poisons NaF and dinitrophenol. This was thought to be due to inhibition of GA metabolism. The accumulation of 3H-GA1in aleurone tissue did not reach saturation when unlabeled GA3 up to 10-31~ was added to the incubation medium.

Introduction

The accumulation of animal hormones in target tissues is a welldocumented phenomenon (see e.g. Jensen and Jacobsen, 1962). Such in rive binding studies have led to the isolation and characterization of hormonal receptor proteins (see e.g. Gorski et al., 1968). The accumulation of plant hormones in their respective target tissues or target cells has been demonstrated in only two cases: Radioactive gibberellins (GAs) accumulate in the apical, GA-sensitive p a r t of the dwarf pea stem (Musgrave et al., 1969) and a labeled eytokinin, benzyladenine, in those cells of moss protonemata which form buds in response to the hormone, and in the developing buds themselves (Brandes and Kende, 1968). The experiments described below were performed in order to study the uptake, accumulation and metabolism of GAs by barley aleurone cells which synthesize a number of hydrolytic enzymes in response to GA. * Abbreviations: GA, gibberellin; GAs-ME, gibberellin A5 methyl ester; RNase, ribonuclease. 1

Planta (Berl.), Bd. 102

2

A. Musgrave, S. E. Kays, and H. Kende: Materials and Methods

Incubation. Aleurone layers of barley (Hordeum vulgate, L., cv. Himalaya, 1964 harvest) were prepared according to Chrispeels and Varner (1967), and incubated at 25~ on a reciprocating shaker in filter-sterilized Na-acetate buffer (0.001 M, pH 4.8) containing 0.01 IV[CaC12and 10 ~g/ml chloramphenieol. Where indicated, the following compounds were included in the incubation medium: 3H-GA1 (spec. activity 48 mc/mmole; Kende, 1967), aH-GA5 (spec. activity 110 mc/mmole; Musgrave and Kende, 1970), 3H-GAs-ME (spec. activity 110me/mmole; Musgrave ctal., 1969) all at a concentration of 2 X 10-7 M, and NaF and dinitrophenol at 10-3 M. Enzyme Assays. At appropriate intervals, samples of the incubation media were removed aseptically, frozen and kept at --20 ~ until further analysis, a-Amylase and RI~ase aet'lvities were measured as described by Chrispeels and Varner (1967). Assay o/ Radioactivity. The aleurone layers were blotted, weighed and shaken overnight in vials containing Bray's (1960) scintillation fluid. The radioactivity was determined with a Packerd Liquid Scintillation Spectrometer (Model 3375). Zones of thin-layer chromatograms were scraped off and counted directly in Bray's solution. When necessary, the results were corrected for quenching. Extraction o] GAs and GA-Metabolites. The layers were removed from the incubation medium and washed for I h in two fresh batches of GA-free medium. The medium and the combined wash solutions were partitioned against ethyl acetate, first at pH 8.5, then at 2.5. The resulting three phases--non-acidic ethyl-acetate, acidic ethyl-acetate and the remaining aqueous phase--were tested separately for radioactivity. The aleurone layers were ground in methanol using a glass mortar and pestle. The mixture was centrifuged, the methanol decanted, and the residue extracted twice more with methanol. The methanol extracts were combined, and the solvent was evaporated under vacuum. The residue was taken up in ethyl acetate and phosphate buffer at pH 8.4 and partitioned. The pH of the aqueous phase was adjusted to 2.5 with HC1 and partitioning against ethyl acetate was repeated. The radioactivity of the non-acidic ethyl-acetate, acidic ethyl-acetate and aqueous phases was determined. The methanol-insoluble residue was dried, combusted in a Packard Sample Oxidizer and the radioactivity of the resulting tritiated water was counted. Chromatography. The relative distribution of GAs and GA-metabolites in the 2 ethyl-acetate phases was determined by thin-layer chromatography using Silica gel t t and the following solvent systems: A. benzene--n-butanol--acetic acid (70: 20: 2, v/v), B. chloroform--ethyl acetate--acetic acid (60: 40: 5, v/v).

Results G A 1 and, to a lesser e x t e n t , G A 5 e n h a n c e d t h e p r o d u c t i o n of ~ - a m y lase in i s o l a t e d aleurone layers (Fig. 1). T h e earliest effect of GA1, like t h a t of GAa, was m e a s u r a b l e after 7-8 h, while t h e earliest response to G A 5 could be d e t e c t e d o n l y a f t e r 12 h. GAs-ME d i d n o t enhance t h e f o r m a t i o n of ~-amylase. T h e level of t h e e n z y m e in control a n d GAs-MEt r e a t e d aleurone layers increased slowly beginning a b o u t 15 h after t h e s t a r t of i n c u b a t i o n . Alcurone l a y e r s i n c u b a t e d in G A 1 solution a t 4 ~ m a i n t a i n e d a w a t e r c o n t e n t of 50 % b y weight, whereas t h e w a t e r c o n t e n t of layers k e p t a t 25 ~ rose to 80% over a p e r i o d of 50 h. If t h e w a t e r in t h e tissue h a d

Uptake and Metabolism of Gibberellins

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Fig. 1. Secretion of ~-amylase in response to GA treatment. 20 aleurone layers were incubated in 300 ml Erlenmeyer flasks at 25~ in 10 ml medium with or without 2 • 10-TM3H-GA1,3H-GAs or 3H-GAs-ME. At intervals, 0.1-ml samples were taken from the medium and kept frozen at --20 ~ until assayed for ~-amylase

equilibrated with the external solution containing 2 • 10-~M ~H-GA~, the level of radioactivity would have varied from 35 cpm/10 mg fresh weight at 50 % water content to 50 cpm/10 mg at 80 % water content. Fig. 2 shows t h a t aleurone layers kept at 4 ~ equilibrated with the external solution while layers at 25 ~ accumulated radioactivity for 25 h. At this point, accumulation stopped and radioactivity was released into the medium. I n a few experiments there was an initial equilibration at 50 epm/10 mg with a later, second phase of accumulation about 8 h after the start of incubation. This pattern was characteristic for 8H-GA 5 and 3H-GAs-ME (Fig. 4). Aleurone layers treated with 8It-GA1 and N a F or dinitrophenol at 25 ~ did not accumulate radioactivity but nevertheless took up some more 8H-GA1 than aleurone layers without inhibitors at 4 ~ (Fig. 2). During the period when aleurone layers were capable of accumulating labeled GA and its metabolites, e.g. after 9 and 22 h of incubation, they strongly retained the accumulated radioactivity. When the tissue was washed in GA-free medium, some exchangeable counts leached out immediately, but the greater p a r t of the radioactivity was lost only slowly to the external medium (Fig. 3). I n contrast, tissue t h a t had been incubated in 8H-GA1 for 40 h and had lost the ability to accumulate radioactivity had also lost the ability to retain it. Washing this tissue removed all counts except a residual level of approximately 10 epm/10mg which could be attributed to insoluble metabolites. I f the accumulation of radioactivity was due to 3H-GA1 being bound to a finite number of specific sites, these should become saturated at a 1"

4

A. Musgrave, S. E. Kays, and H. Kende:

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Incubation Time (hr.) Fig. 2. Levels of radioactivity in aleurone layers incubated in aH-GAz solution. 50 aleurone layers were incubated in 500 ml Erlenmeyer flasks at 25~ or 4~ in 20 ml medium containing 2 X 10-rIVISH-GA1. At intervals, 3-5 layers were removed, weighed, and assayed for radioactivity. Some layers were first incubated at 4~ and later (see arrow) transferred to 25~. Other layers were incubated in the presence of NaF or dinitrophenol, both at 10-aM. Since the uptake of radioactivity was identical in the presence of dinitrophenol and NaF, only the results with NaF are shown Table 1. Competition between aH-GA1 and GA s 10 aleurone layers were incubated in 10 ml medium in 150 ml Erlenmeyer flasks. The medium contained 2 • 10-TM SH-GA1 and different concentrations of GAs. After incubating for 21 h, 5 layers were assayed for radioactivity. The remaining 5 layers were washed in GA-free medium for 4 h after which they were assayed for radioactivity. GAa concerttration (M) 0 10 -s

10-5 10-a 10-a 10-2

Radioactivity (cpm/10 mg fresh weight) Mter 21 h

After 4-h wash

123 141 129 120 129 127

83 102 91 85 69 85

certain GA c o n c e n t r a t i o n above which no f u r t h e r a c c u m u l a t i o n would be expected. Because of the limited supply of SH-GA1 a n d GA1, commercially available GAs was used i n competition experiments with a l l - G A 1. These 2 GAs are chemically closely related a n d have the same s p e c t r u m of biological activity. Aleurone layers were i n c u b a t e d i n 2 x 10-7 M

Uptake and Mef~bolism of Gibberellins

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Fig. 3. l~elease of radioactivity from cells incubated in att-GA r Aleuronc layers were incubated in medium containing 2 • 1 0 - ~ 3H-GAz for 9, 22, or 40 h. At these times, some were assayed for radioactivity while others were blotted and transferred to GA-free medium. At intervals, 3-4 layers were removed from the washing medium and assayed for radioactivity

3H-GA1 to which GA 3 was added at concentrations up to 10 -2 M. The radioactivity of the aleurone layers was assayed after 21 h of incubation and after an additional washing period of 4 h. As shown in Table 1, it was impossible to demonstrate a saturation of binding sites with GA s at concentrations as high as 10-2 M. Fig. 4 illustrates the uptake and accumulation of 3H-GAs and ~I-I-GA~-ME b y aleurone layers. If the water in the tissue had equilibrated with the external solution, the level of radioactivity would have varied from 80 to 115 cpm/10 mg fresh weight, depending on water content. These figures are higher than those for 3H-GA1 because the specific activity of 3tI-GA 5 was correspondingly greater. Comparing peak levels of accumulated radioactivity, 3H-GAl-treated cells accumulated twice the background level while tissue treated with 8H-GAs and 3H-GAs-ME accumulated 15 and 34 times the background level, respectively. At this point, we investigated whether aleurone layers accumulated unchanged GA or GA-metabolitcs. Aleurone layers were incubated in ~H-GA 1 for 20 h and in 3H-GAs and aH-GA6-ME for 24 h. Thereafter, they were blotted and washed for 1 h in GA-free medium to remove freely exchangeable counts. Table 2 shows the distribution of radioactivity in the incubation media, the washings, and the methanol extracts of the tissue for each GA treatment. By comparing the total radioactivity in the tissue with the total radioactivity in each incubation flask, it was calculated t h a t aleurone layers treated with aI-I-GA1 accumulated only 4% of the originally added counts while aleurone layers

6

A. Musgrave, S. E. Kays, and H. Kende:

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Fig. 4. Levels of radioactivity in aleurone layers incubated in 3H-GA5 or 3H-GAs-ME. 50 aleurone layers were incubated in 500 ml Erlenmeyer flasks at 25~ in 20 ml medium containing 2 • 10-vM 3H-GAs or 3I-I-GA5-ME. At the indicated times, 3~5 layers were removed and assayed for radioactivity

treated with 3H-GAs and 3It-GAs-ME accumulated 18 and 22%, respectively. If there had been no metabolism of applied GA, 3H-GA1 and aH-GA 5 would have partitioned into the acidic and 3H-GA5-ME into the non-acidic ethyl-acetate phase. I n all treatments most of the radioactivity extracted from aleurone layers remained in the aqueous phase and was evidently associated with polar substances. These polar compounds, rather than unmetabolized GA, thus represent the bulk of the radioactivity accumulated in the tissue. Thin-layer chromatography was used to determine the amount of unmetabolized GA (Fig.5) in the ethyl-acetate phases. The chromatograms of the water washes are not shown because they were very similar to those of the incubation media. When the tissue extracts were analysed, it was found t h a t less t h a n 1% of the radioactivity accumulated was unmetabolized aH-GA~ and 3H-GAs-ME whereas unchanged aH-GA1 accounted for 15% of the accumulated counts in the respective treatments (Fig. 5 d - f ; Table 2). The major radioactive component remained at the origin of the ehromatograms, indicating t h a t it was of polar nature. We did not determine whether these polar compounds at the origin of the chromatograms were identical with those in the aqueous phase. I n the incubation media, nearly all the radioactivity remained associated with unmetabolized GA 1 and GA5, respectively (Fig. 5a, b;

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Non-acidic ethyl acetate Acidic ethyl acetate Aqueous Methanol-insoluble residue Unmctabotized GA Total radioactivity (cpm)

X~r~ction

0 82.0 18.0 - -

82.0 2199

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15.2 4510

8 . 9

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Medium

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Table 2. Metabolism o/radioactive gibberellins 50 aleurone layers were i n c u b a t e d for 20 h in 2 x 10-7 ~ aH-GA1 or for 24 h in 2 • 10 -v M all-GAs or all-GAs-ME. The layers were t h e n removed from the incubation media, blotted, a n d washed for 1 h with 2 lots of GA-free medium. T h e distribution of radioactivity was expressed as percentage of t h e t o t a l cpm in each fraction.

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Uptake and Metabolism of Gibberellins

9

Table 2). In the case of 3tLGAs-ME, only 27% of the original GA was unmetabolized (Table 2; Fig. 5 c). The remaining 73 % were in the form of more polar metabolites but without a major component remaining at the origin of the chrom~togram (Fig. 5 @ This may imply that not all metabolites were accumulated but only the strongly polar ones; others, like the parent gibberellins, exchanged apparently quite freely between tissue and medium. The distribution of radioactivity in the washings was similar to the distribution of radioactivity in the external solution, except for a higher proportion of counts remaining in the aqueous phase (Table 2). We presume that this was because a small proportion of the polar material leached from the tissue during the 1-h wash. After 20-30 h of incubation, the accumulated radioactivity was released from the tissue. At the same time, RNase was also liberated into the medium. This correlation is shown in Fig. 6 for aleurone layers which had been treated with 8H-GAr Also shown is the release of RNase from untreated aleurone layers. In the experiment of Fig. 6, the radioactivity and RNase were liberated after 21 h of incubation. In other experiments, an equally well correlated release of SH-GA5 and RIqase was observed after 28 h of hormone treatment. Discussion

In earlier work (Musgrave eta[., 1969), we were able to demonstrate accumulation of radioactive GAs in the growing, GA-sensitive region of the pea stem. When seedlings were treated with biologically inactive GA derivatives, such as the methyl ester of 3H- GA 5, very little or no accumulation was found. Based on such correlations, we proposed that the accumulation of GA in pea apices was connected with the action of the hormone. In aleurone layers, which also represent a target tissue for GA, we were unable to show a positive correlation between GA accumulation and biological activity of the hormone. In fact, the degree of accumulation and the biological activity of the GA were related inversely. From the metabolic studies it became evident that the accumulated radioactivity was primarily associated with polar metabolites of the respective GA. The extent of accumulation was proportional to the degree of metabolism of the GAs used (GA~-ME ~ GA 5 ~ GAs). No accumulation was observed at 4 ~ probably because GA was not metabolized at this temperature. In the presence of NaF or dinitrophenol at 25 ~ a slight accumulation of label was noted (Fig. 2), possibly due to GA metabolism supported by residual ATP. In earlier, preliminary experiments (Kende, 1967), no significant metabolism of 3H-GA~ had been detected in aleurone layers. In those experiments, the aleurone layers and the incubation medium were

10

A. Mnsgrave et al. Uptake and Metabolism of Gibberellins

extracted together. Since the aleurone layers metabolize about 5% of the total applied GA1, this relatively small fraction of metabolites appears to have been masked b y the bulk of the unmetabolized hormone. Polar GA metabolites were held back in the cell and were not free to diffuse from the tissue into the m e d i u m for the first 20-30 h of incubation. The simultaneous release of GA metabolites and R N a s e suggest the desintegration of some cellular compartment(s) at this particular physiological stage. This work was supported by the U.S. Atomic Energy Commission under Contract No. AT(11-1)-1338 and by the National Science Foundation (Grant I~o. GB-13853). References Brandes, H., Kende, H. : Studies on cytokinin-controlled bud formation in moss protonemata. Plant Physiol. 48, 827-837 (1968). Bray, G. A. : A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, 279-285 (1960). Chrispcels, M. J., Varner, J. E. : GibbereUic acid-enhanced synthesis and release of s-amylase and ribonuelease by isolated barley aleurone layers. Plant Physiol. 42, 398-406 (1967). Gorski, J., Tof% D., Shyamala, G., Smith, D., ~qotides, A.: Hormone receptors: Studies on the interaction of estrogen with the uterus. Recent Progr. Hormone Res. 24, 45--80 (1968). Jensen, E. V., Jacobson, H. J. : Basic guides to the mechanism of estrogen action. Recent Progr. Hormone Res. 18, 387-414 (1962). Kende, H. : Preparation of radioactive gibberellin A 1 and its metabolism in dwarf peas. Plant Physiol. 42, 1612-1618 (1967). Musgrave, A., Kende, H. : Radioactive gibberellin A5 and its metabolism in dwarf peas. Plant Physiol. 45, 53-55 (1970). - - Kays, S. E., Kende, H. : In-vivo binding of radioactive gibberellins in dwarf pea shoots. Planta (BEE.) 89, 165-177 (1969). Alan Musgrave Unit of Developmental Botany Agricultural Research Council University of Cambridge Cambridge CB30DY, U.K.

Hans Kende MSU/AEC Plant Research Laboratory Michigan State University East Lansing, Michigan 48823 U.S.A.