Epinephrine, Propranolol, and the Sucrose-Ammonium Inhibition of ...

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Jan 13, 1982 - JANET M. IVES2 AND HERBERT B. POSNER3. Department ofBiological ... 'Dedicated to the memory of Dr. William S. Hillman. 2 Present address: Department of Biology, University of Rochester,. River Campus Station ...
Plant Physiol. (1982) 70, 311-312 0032-0889/82/70/031 1/02/$00.50/0

Epinephrine, Propranolol, and the Sucrose-Ammonium Inhibition of Flowering in Lemna paucicostata 67461 Received for publication September 9, 1981 and in revised form January 13, 1982

JANET M. IVES2 AND HERBERT B. POSNER3 Department of Biological Sciences, State University of New York, Binghamton, New York 13901 medium with 1% (w/v) sucrose (pH 6.4) in 125-ml Erlenmeyer flasks under 2,000 to 2,500 lux of continuous cool-white fluorescent light at approximately 25°C (10). Experimental cultures, started with one three-frond colony on 20 ml medium in 25 x 150 mm tubes with stainless steel closures, were grown for 8 or 9 d under continuous blue fluorescent light filtered through 3 mm of Rohm and Haas #2045 blue Plexiglas. The irradiance was 800 ergs cm-2 s-5. We have found that these growth conditions (3) gave a more reliable and reproducible flowering response to ammonium ion than did those used previously (4, 11). However, as noted below, the response was also determined under short days. The experimental medium was half-strength Hutner medium with sucrose as described above or the ammonium-free modification in which equimolar KNO3 substituted for the NH4NO3. Sterilization was by autoclaving except for L-epinephrine, DLpropranolol (1-[isopropylaminoJ-3-[1-naphthyloxylJ-2-propanol) and cAMP (all from Sigma Chemical Co.) which were added by sterile filtration. There were four or more cultures for each treatment. Flowering intensity is expressed as the mean FM%. The latter is the number of flowering fronds divided by the total number of visible fronds and multiplied by 100. Statistical treatment of data involved analysis of variance and Duncan's new multiple range test as described by Steel and Tome (14).

ABSTRACT The sucrose-ammonium inhibition of flowering Lemnapaucicostata 6746 in continuous blue light or in short days was partially overcome by epinephrine. This reversal was prevented by propranolol, an antagonist of epinephrine in animals. In ammonium-free medium, propranolol inhibited flowering, and this inhibition was completely overcome by epinephrine. Increased levels of Ca2", Pi and nitrate partially reversed the inhibition by propranolol. Concentrations of cAMP, adenine, and adenosine that partially overcame the sucrose-ammonium inhibition did not affect flowering in cultures treated with propranolol. The possibility is discussed that the effects on flowering of sucrose-ammonium, propranolol, and epinephrine were due to altered intracellular levels of cAMP or of a cAMP-like compound.

Inhibition of flowering by ammonium ion and by sucrose has been observed in several species of Lemnaceae (5, 6, 16). In the SDP4 Lemna paucicostata5 strain 6746, the sucrose inhibition (11) was later shown to be due to an interaction between sucrose and ammonium ion (4). Oota (6, 8) reported that inhibition of flowering by either sucrose or ammonium ion in the LDP L. gibba G3 was reversed RESULTS by cAMP. The inhibition by sucrose was partially reversed by catecholamines such as epinephrine which, in animals, increase Initial experiments tested the effects of epinephrine on flowering intracellular levels of cAMP. Further, the reversal was prevented in the absence and presence of ammonium ion. At both concenby propranolol, an antagonist of epinephrine. On the basis of trations tested, 10 and 50 IsM, epinephrine had no effect on these and other results, Oota (7) proposed that the inhibition of flowering in ammonium-free medium, but did significantly inflowering in L. gibba G3 was due to reduced levels of intracellular crease F 1% in medium containing ammonium ion (Table I). cAMP. Intracellular levels of cAMP have been shown to be The ability of epinephrine to reverse at least partially the lowered by sugars in certain bacteria (9) and by ammonia in slime inhibition by ammonium ion was prevented by propranolol (Table mold (13). II). Further, on ammonium-free medium, propranolol inhibited The purpose of the following study was to determine whether flowering (Table III), and as in the case of the ammonium flowering responses of L. paucicostata 6746 to epinephrine and inhibition, the inhibition by propranolol was reversed by epinephpropranolol were consistent with a possible involvement of cAMP in the sucrose-ammonium inhibition. Table I. Effects of Epinephrine on Ammonium Inhibition of Flowering Cultures were grown for 9 d on ammonium-containing half-strength MATERIALS AND METHODS Hutner medium with 1% sucrose (+NH4+) or its ammonium-free modifiVegetative stock cultures of Lemna paucicostata 6746 were cation (-NH4+) in continuous blue light. Epinephrine was added by sterile grown axenically on approximately 50 ml half-strength Hutner filtration. Values followed by the same letter are not significantly different at the 1% probability level.

'Dedicated to the memory of Dr. William S. Hillman. 2 Present address: Department of Biology, University of Rochester,

Fl%

Epinephrine

River Campus Station, Rochester, NY 14627. 3To whom correspondence should be addressed. 4Abbreviations: SDP, short-day plant; LDP, long-day plant; FM%, flowering percent. 5This clone was formerly designated as Lemna perpusilla, but was later identified as Lemna paucicostata.

0 10 50

311

-NH4+

+NH4+

48.0 A 53.7 A 49.6 A

2.7 B 15.4 C 21.2 C

IVES AND POSNER

312

Table II. Effects of Propranolol on Epinephrine Reversal of SucroseAmmonium Inhibition of Flowering Cultures grown in ammonium-containing half-strength Hutner medium as in Table I. Propranolol concentration was 50 MM. Fl% Epinephrine +Propranolol -Propranolol M 1.5 A 5.7 A 0 4.5 A 18.4B 10 1.6 A 28.8 C 50 Table III. Effects of Epinephrine on Propranolol Inhibition of Flowering Cultures grown in ammonium-free modification of half-strength Hutner medium with 1% sucrose as in Table I. Epinephrine concentration was 50

Plant Physiol. Vol. 70, 1982

flowering of L. paucicostata 6746. Propranolol, an antagonist of epinephrine in animals, inhibited flowering of Lemna in ammonium-free medium. As in the case of the sucrose-ammonium inhibition (1 1, 12), the propranolol inhibition of flowering was at least partially reversed by epinephrine and by increased levels of

Ca2" and Pi.

Some of these effects of epinephrine and propranolol on flow-

ering have been reported for the LDP L. gibba G3 (7). On the basis of such results, Oota (6-8) suggested that inhibition of flowering by sugar and ammonium ion was due to lowered levels of cAMP. There is, however, no evidence that the effects of epinephrine and propranolol in plants involve mechanisms similar to those in animals. In fact, there is not complete agreement concerning the existence of cAMP in plants (1), althougb) recent

evidence suggests that some higher plants synthesize cAMP (see, e.g. 2) or some closely related compound (15). Oota (8) showed that supplementing the medium with low MM. concentrations of cAMP reversed the ammonium inhibition of flowering of L. gibba G3, whereas other adenylates were ineffecF1% tive. In contrast, reversal of the sucrose-ammonium inhibition in Propranolol L. paucicostata required high concentrations of cAMP, and other +Epinephrine -Epinephrine adenylates (12) as well as adenine and adenosine were at least MM equally effective. However, these compounds had no effect on the 45.5 A 48.1 A 0 inhibition by propranolol. Taken together, these findings suggest 44.4 A 13.3 B 50 that the mechanism of action for sucrose-ammonium inhibition differs from that for propranolol, and that in L. paucicostata the Table IV. Effects of Various Salts on Propranolol Inhibition of Flowering effects of exogenous cAMP and other adenine-containing comThe basal medium was ammonium-free half-strength Hutner medium pounds might be due to their conversion to an active derivative. (1% sucrose) with or without propranolol (50 Mm). The first four salts listed were added so txat their concentrations were those of full-strength medium. Other conditions as in Table I.

Acknowledgment-We thank Arthur H. Levine for technical assistance.

F1%

LITERATURE CITED

Supplement -Propranolol

+Propranolol

mM

None

K2HPO4 (1.15) MgSO4 (1.00) Ca(NO3)2 (0.75) KNO3 (1.25) KNO3 (1.50) CaCl2 (0.75) KC1(1.50)

49.1 A 47.4 A 47.1 A 51.0 A 52.2 A 47.0 A 46.5 A 46.5 A

15.2 B 31.8 C 12.9 B 35.8 C 30.3 C 26.8 C 33.1 C 13.4 B

rine. This reversal, however, seemed to be complete. Similar results were obtained under short days on tenth-strength Hutner medium with sucrose. Earlier results (11) showed that raising the level of Ca2" or Pi partially overcame the sucrose-ammonium inhibition. The results in Table IV indicate that the inhibition by propranolol was also partially overcome by increased levels of these ions, but nitrate also was effective. Inasmuch as high concentrations of cAMP were effective in partially overcoming the sucrose-ammonium inhibition (12), it was of interest to determine whether the inhibition by propranolol was similarly affected. We confirmed partial reversal of the sucrose-ammonium inhibition by cAMP (300 AM) and also found adenine (400 AM) and adenosine (800 Mm) caused reversals. However, these supplements had no effect on the inhibition of flowering by 50 AM propranolol. DISCUSSION

Epinephrine, which is known to increase cAMP levels in animals, partially overcame the sucrose-ammonium inhibition of

1. AMRHEIN N 1977 The current status of cyclic AMP in higher plants. Annu Rev Plant Physiol 28: 123-132 2. BROWN EG, T AL-NAJAFI, RP NEWTON 1979 Adenosine 3':5'-cyclic monophosphate, adenylate cyclase and a cyclic AMP binding-protein in Phaseolus vulgaris. Phytochemistry 18: 9-14 3. HILLMAN WS 1967 Red light, blue light and copper ion in the photoperiodic control of flowering in Lemna perpusilla 6746. Plant Cell Physiol 6: 499-506 4. HILLMAN WS, HB POSNER 1971 Ammonium ion and the flowering of Lemna perpusilla. Plant Physiol 47: 586-587 5. KANDELER R 1969 Hemmung der Blutenbildung von Lemna gibba durch Ammonium. Planta 84: 279-291 6. OOTA Y 1972 A possible mechanism for sugar inhibition of duckweed flowering. Plant Cell Physiol 13: 195-199 7. OOTA Y 1974 Removal by catecholamines of sugar inhibition of flowering in Lemna gibba G3. Plant Cell Physiol 15: 63-68 8. OOTA Y, T KONDO 1974 Removal by cyclic AMP of the inhibition of duckweed flowering due to ammonium and water-treatment. Plant Cell Physiol 15: 403411 9. PASTAN I, R PERLMAN 1970 Cyclic adenosine monophosphate in bacteria. Science 169: 339-344 10. POSNER HB 1967 Aquatic vascular plants. In F Wilt, N Wessels, eds, Methods in Developmental Biology. Thomas Y Crowell Company, New York, pp 301-

317

11. POSNER HB 1969 Inhibitory effect of carbohydrate on flowering in Lemna perpusilla. I. Interaction of sucrose with calcium and phosphate ions. Plant Physiol 44: 562-566 12. POSNER HB 1973 Reversal of sucrose inhibition of Lemna flowering by adenine derivatives. Plant Cell Physiol 14: 1199-1200 13. SCHINDLER J, M SUSSMAN 1979 Inhibition by ammonia of intracellular cAMP accumulation in Dictyostelium discoideum: its significance for the regulation of morphogenesis. Dev Gen 1: 13-20 14. STEEL RGD, JH TORRIE 1960 Principles and Procedures of Statistics. McGrawHill, New York, pp 107-109 15. WOOD HN, AH POMERANTZ, AN BINNS, VG ALLFREY, AC BRAUN 1978 Inhibition of mammalian protein kinase and phosphodiesterase activities by a cyclic AMP-like compound isolated from higher plants. Proc Natl Acad Sci USA 75: 4301-4305 16. YUKAWA I, A TAKIMOTO 1976 Flowering responses of Lemna paucicostata in Japan. Bot Mag Tokyo 89: 241-250