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Effects of cytokinins on promotion of cell arrest in G2 by trigonelline and trigonelline concentrations in cultured roots of Pisum sativum and glycine max
EnvironmentalandExperimentalBotany,Vo[. 25, No. 1, pp. 8348, 1985.
0098~472/85 $3.00 + 0.00 ~ 1985 Pergamon Press Ltd.
Printed in Great Britain.
EFFECTS OF C Y T O K I N I N S O N P R O M O T I O N OF CELL A R R E S T IN G2 BY T R I G O N E L L I N E A N D T R I G O N E L L I N E C O N C E N T R A T I O N S IN C U L T U R E D R O O T S OF PISUM SATIVUM A N D GLYCINE M A X A. T R A M O N T A N O , LANCE 8. EVANS a n d PAULA A. M C G I N I ~ Y Laboratory of Plant Morphogenesis, Manhattan College, The Bronx, NY 10471, U.S.A.
(Received 3 July 1984; accepted in revisedform 20 August 1984) TRAMONTANOW. A., EVANSL. S. and McGINLEY P. A. Effects ofcytokinins onpromotion of cell arrest by trigonelline and trigonelline concentrations in cultured roots of Pisum sativum and Glycine max. ENVmONMENTAL ,',NO EXPERXMENTAL BOTANY 25~ 83--88, 1985.--Effects of cytokinins on trigonelline's promotion of cell arrest in G2 were determined. Previous results demonstrate that trigonelline, (N-methyl nicotinic acid), a hormone present in cotyledons of young seedlings of Pisum sativum L. and Glycine max Merrill, is transported to roots and shoots where it stops cell progression in the G2 stage of the cell cycle during normal cell maturation. In P. sativum, two natural cytokinins, isopentenyl adenine and zeatin, and both their ribosides (all at 10-7 M) were antagonistic to trigonelline's function when placed individually in aseptic culture with trigonelline at 10- 6 M. Elevated trigonelline concentrations ( 10- 4 M) negated the antagonistic effect on these cytokinins. Kinetin and benzyl adenine (synthetic cytokinins) were not antagonistic to trigonelline at any concentration. In G. max, two natural cytokinins, isopentenyl adenine and zeatin, at 10 -7 and 10 -8 M, promoted cell arrest in G2 in the absence of trigonelline. Moreover, the proportion of cells arrested in G2 was higher in roots cultured with isopentenyl adenine in combination with trigonelline, than with either hormone alone. In both legumes, these natural cytokinins did not influence trigonelline concentrations in roots. These results demonstrate an interaction between natural cytokinins and trigonelline in control of the cell cycle in peas and soybeans. This is the first report of an interaction of two naturally occurring hormones that affect cell arrest in either G1 or G2 in tissues of either plants or animals.
INTRODUCTION
tents (in G1 a n d G2, respectively) in s t a t i o n a r y phase meristems are similar to p r o p o r t i o n s in m o r e m a t u r e root segments. ¢5) Few cells arrest d u r i n g D N A synthesis a n d no cells arrest d u r i n g mitosis. ¢5) U n d e r t e m p o r a r y c a r b o h y d r a t e deprivation, root cells o f P . sativum arrest in G 1, in G2, or become p o l y p l o i d J 1n) I f trigonelline is present, as m u c h as 6 0 % of the meristem cell p o p u l a t i o n arrests in G 2 J 1) I f sufficient trigonelline is not present, these cells arrest only in G1. ~1'1°'14) O t h e r n a t u r a l l y occurring p l a n t h o r m o n e s such as zeatin a n d isopentenyl a d e n i n e a n d their ribosides belong to a g r o u p of p h y t o h o r m o n e s called cytokinins. T h e first observed effect of cytokinins as a p l a n t h o r m o n e was the stimulation of cell division in tobacco pith callus. ~s'12) Even
TRIGONELLINE functions as a p l a n t h o r m o n e in Pisum sativum. ¢2"3'1°) Trigonelline, found in cotyledons of d r y seeds, ¢4) is t r a n s p o r t e d from cotyledons to other p l a n t tissues d u r i n g early seedling d e v e l o p m e n t ~13) a n d promotes preferential cell arrest in G2 of the cell cycle. ~2'4) Arrest of meristematic cells in the cell cycle in cultured p r i m a r y root tips after t e m p o r a r y c a r b o h y d r a t e d e p r i v a t i o n (to p r o d u c e s t a t i o n a r y phase meristems) is a n o n - r a n d o m process. Cx) Cell arrest d u r i n g n o r m a l cell differentiation (e.g. in m o r e m a t u r e segments) also occurs as a n o n - r a n d o m process. ~) Results have also shown t h a t the proportions of cells with 2C a n d 4C D N A con83
84
W.A. TRAMONTANO et al.
today, cell division activity is the most frequently observed result of cytokinins. ~6'16) Research on the control of cell division by cytokinins has occurred with individual cells and callus in aseptic cultureJ 16) The results described below point to a mechanism for control of cell proliferation in a complex tissue. The mechanism postulated herein is an interaction of cytokinins with trigonelline. Two experimental procedures were performed to determine the interactive effects of trigonelline and cytokinins. Experiments were performed to determine if cytokinins influence cell arrest in G2 alone and in combination with an effective trigonelline concentration. Since four natural cytokinins were shown herein to affect trigonelline's function, experiments were performed to determine if one of these four cytokinins (isopentenyl adenine) affected trigonelline concentrations in excised roots.
MATERIALS AND M E T H O D S
Bioassayfor promotion of cell arrest in G2 The general methods used in the bioassay have been described elsewhere, m Seeds ofPisum sativum L. and Glycine max Merrill were surface sterilized with undiluted Clorox® for 15 min, washed with sterile water to remove bleach, germinated in sterile Vermiculite®, and grown for 3 days. Under sterile conditions, excised root tips were placed in White's medium with sucrose and with various concentrations of cytokinins (Sigma Chemical Co., St. Louis, MO), with and without trigonelline. Appropriate medium controls were also used. Initially, six cytokinins at three different concentrations (10 -6, 10 .7 and 10 .8 M) were tested either with or without 10 -6 M trigonelline. Of the cytokinins tested, four were natural--isopentenyl adenine (IPA), isopentenyl adenine riboside, zeatin, and zeatin riboside. Two synthetic cytokinins, kinetin and benzyl adenine were also tested. After 3 days, root meristems were transferred to White's medium without carbohydrate or any of the test chemicals for 4 days so that cells were present only in the G1 and G2 stages of the cell cycle. The procedure used for soybeans was identical, except that seedlings were grown for 4 days before excision and the cyto-
kinins tested were IPA and zeatin at concentrations of l 0 - 7 and 10- s M. After carbohydrate deprivation, roots were fixed in a mixture of ethanol and acetic acid (3 : 1 by volume). Relative amounts of DNA per nucleus were determined on Feulgen stained nuclei from 0 to 2 mm terminal meristems by microfluorimetry. Measurements of nuclei were normalized with readings of one-half telophase and prophase figures taken to be 2C (G 1) and 4C (G2) values, respectively. (1)
Trigonelline concentrations To determine effects of cytokinins on trigonelline concentrations in cultured roots ofP. sativum and G. max, seeds were surface sterilized, washed with sterile water, germinated in sterile Vermiculite®, and grown for 3 and 4 days, respectively. Under sterile conditions, excised root tips were placed in medium with sucrose and IPA at concentrations of 10-v and 10 .8 M, with and without 10 .6 M trigonelline. Appropriate medium controls were used. After 3 days, roots were harvested and 0 1 cm terminal root segments were excised and extracted in an ethanol series. ~7,14~ After three extractions with chloroform to remove lipids, each ethanol extract was concentrated, spotted in Analtech silica gel thinlayer chromatography u.v. plates, 250 ~tm in thickness (Anahech Corp., Newark, DE 19711), and developed in acetone:water (50:50, v/v). Plates were allowed to air dry and trigonelline was eluted. Trigonelline concentrations were determined using high-pressure liquid chromatography. ~11'14) A Dupont Liquid Chromatograph Model 830 with an Ultraviolet (254 nm) detector and a Permaphase C-18 ODS column was used. For HPLC, methanol : water (50 : 50) was used as a solvent at a flow rate of 1 ml/min. ~14~ RESULTS Effects of added cytokinins on promotion of cell arrest in cultured roots ofP. sativum are shown in Table 1. The normal range for stationary phase meristems ofP. sativum is between 0.1 and 0.2 for cells in G2J 1) Cytokinins had no significant influence on the proportion of cells arrested in G2 in the absence of trigonelline. Isopentenyl adenine, trans-zeatin, and their ribosides, separately
EFFECTS OF C Y T O K I N I N S IN ROOTS OF P. S A T I V U M
85
Table 1. Antagonistic effects of natural cytokinins on promotion of cell arrest in G2 in excised roots of Pisum sativum
Concentration of cytokinin (M) 10 .7 M
10-aM
Isopentenyl adenine Isopentenyl adenine riboside Zeatin Zeatin riboside Kinetin Benzyl adenine Isopentenyl adenine Isopentenyl adenine riboside Zeatin Zeatin riboside Kinetin Benzyl adenine
None
Proportion of cells in G2 Trigonelline concentration (M) 0 10 -6 M 10 -4 M 0.17±0.05" 0.16+_0.03 0.14+_0.03 0.20+-0.02 0.17 ± 0.04 0.11 -t-0.03 0.11 +_0.01 0.13+0.03 0.11+_0.02 0.13+-0.04 0.13+-0.03 0.10+-0.02 0.10-t-0.02
0.17__.0.02 0.13±0.02 0.13+0.03 0.18__.0.03 0.38 +_0.04 0.31 +_0.04 0.30±0.06 0.14+-0.04 0.33+0.04 0.35_+0.01 0.34-t-0.02 0.32+-0.02 0.32+_0.02
0.23-t-0.03 0.39+0.03 0.34+0.03 0.31 -t-0.02 -------
* Mean+S.E. of 3 slides, 50 nuclei were scored on each slide. Each treatment was repeated three times.
at a c o n c e n t r a t i o n of 10-7 M, were antagonistic to trigonelline (10 - 6 M). I n contrast to the response of these n a t u r a l cytokinins, two synthetic cytokinins, kinetin a n d benzyl adenine, were not antagonistic to trigonelline at a n y c o n c e n t r a t i o n tested. A high trigonelline c o n c e n t r a t i o n (10 - 4 M) nullified the antagonistic effects of these n a t u r a l cytokinins at 10 - 7 M (except isopentenyl adenine). M o r e o v e r , no n a t u r a l cytokinin tested (10 - s M), except isopentenyl adenine riboside, was antagonistic to trigonelline (10 - 6 M). I n general, elevated concentrations o f trigonelline n e g a t e d the antagonistic effects of these n a t u r a l cytokinins. Cytokinins at a c o n c e n t r a t i o n of 10-6 M were not tested because they inhibited root growth, with or w i t h o u t trigonelline. These resuits d e m o n s t r a t e that the relative concentrations of trigonelline a n d n a t u r a l cytokinins in aseptic m e d i a m a y d e t e r m i n e w h e t h e r p r o m o t i o n of cell arrest in G2 in P. sativum occurs. Trigonelline concentrations within cultured roots o f P. sativum are shown in T a b l e 2. A n effective c o n c e n t r a t i o n of trigonelline was present in roots exposed to 10 - 6 M trigonelline alone (57.8 #g/g tissue). Those exposed to 10 - 6 M trigonelline with I P A (10 - v or 10 - 8 M; 52.1 and
55.2 #g/g tissue, respectively). I n contrast, when roots were exposed to 10 - 4 M trigonelline a n d 10 -T M I P A , the trigonelline concentration in roots was 390.2 #g/g tissue, far above the effective concentration for p r o m o t i n g cell arrest in G2. These d a t a suggest that the antagonistic effects
Table 2. Concentrations of trigonelline (#g/g tissue) in excised roots of garden peas (Pisum sativum) cultured for 3 days in medium with various constituents added
Treatment Control Trigonelline ( 10- 6 M) Isopentenyl adenine (10- v M) Isopentenyl adenine (10- 7 M) and trigonelline (10 -6 M) Isopentenyl adenine (10 .8 M) Isopentenyl adenine ( 10 -8 M) and trigonelline (10 -6 M)
Trigonelline cone, (/.tg/g tissue) 25.4±6.5* 57.8±6.9 37.6±3.9 52.1 +_ 10.0 29.9+ 1,0 55.2+ 10.2
* Mean+S.E. of 8 experiments in which separate sets of roots were extracted.
86
W. A. TRAMONTANO et al. Table 3. Effects of natural cytokinins on promotion of cell arrest in G2 in excised roots of
Glycine max Concentration of cytokinin (M) 10-7 M 10-8 M
Isopentenyl adenine Zeatin Isopentenyl adenine Zeatin
None
Proportion of cells in G2 Trigonelline concentration (M) 0 10-6 (M) 0.35+0.02* 0.42 _ 0.05 0.36-t-0.03 0.42 +_0.04 0.22 4- 0.04
0.534-0.02 0.44 4- 0.04 0.49___0.01 0.40 4- 0.04 0.42 _ 0.02
*Mean+S.E. of 3 slides, 50 nuclei were scored on each slide. Each treatment was repeated three times.
of IPA cannot be accounted for by low concentrations of trigonelline within roots of P. sativum. Since synthetic cytokinins had no effect on trigonelline's function in P. sativum, only natural eytokinins were tested in G. max (Table 3). Both zeatin and IPA at 10 -7 and 10 - s M promoted cell arrest in G2 in the absence of trigonelline. Isopentenyl adenine in combination with trigonelline (10 -6 M) resulted in a greater proportion of cells arrested in G2 compared with only one compound present. These results demonstrate that IPA has an additive effect for promoting cell arrest in G2 by trigonelline. Zeatin in combination with trigonelline did not produce this additive effect. Trigonelline concentrations in cultured roots of G. max, were higher than controls when cultured with 10 -6 M trigonelline (Table 4). IPA did Table 4. Concentrationsof trigonelline (#g/g tissue) in excised roots of soybeans (Glycine max) culturedfor 3 days in medium with various constituents added
Trigonelline conc.
Treatment No additives Isopentenyl adenine (10-s M) Trigonelline ( 10- 8 M) Isopentenyl adenine (10-s M) and trigonelline (10 -6 M)
(pg/g tissue) 12.04-2.4" 17.14-6.1 42.14-8.2 35.94-1.0
* Mean 4-S.E. of results of 4 experiments in which separate sets of roots were extracted.
not affect trigonelline concentrations in cultured roots of G. max under any conditions. These results suggest that IPA promotes cell arrest in G2 in G. max independent of root trigonelline concentrations.
DISCUSSION Natural cytokinins affect trigonelline's function both in P. sativum and G. max. In P. sativum, cytokinins have an antagonistic effect on trigonelline's promotion of cell arrest in G2. This antagonism by cytokinins can be overridden by relatively high trigonelline concentrations. In this manner, promotion of cell arrest in G2 may be dependent on relative concentrations of trigonelline and cytokinins. The antagonistic effects of natural cytokinins on trigonelline's function in P. sativum cannot be explained by changes in trigonelline concentrations within roots. These results suggest that the antagonistic effects of cytokinins in P. sativum cannot be explained by regulation of trigonelline metabolism within the pyridine nucleotide pathway. In contrast to the situation with peas, natural cytokinins promoted cell arrest in G2 in G. max. In general, the effects of isopentenyl adenine were additive to those of trigonelline in this species. From the available data it seems that isopentenyl adenine promoted cell arrest in G2 independently of trigonelline in G. max. A difference in function between synthetic and natural cytokinins is demonstrated. Although no
EFFECTS OF CYTOKININS IN ROOTS OF P. S A T I V U M explanation is presently available for this difference, it is possible that the structural specificity ofcytokinins governs the inhibition oftrigonelline function in peas in the same way that the structural specificity of trigonelline is a requirement for promotion of cell arrest in G2. (9) Isopentenyl adenine and zeatin are alike structurally, the only difference between the two compounds is a hydrogen in I P A which is substituted by a hydroxyl group in zeatin. Moreover, zeatin and I P A are similar in function in relation to effects of trigonelline function. Kinetin and benzyl adenine are not similar in structure and function to the two natural cytokinins. It m a y be questioned whether trigonelline and/or natural cytokinins suppress the cell cycle or change the average phase durations. From available evidence (1, unpublished results) it seems that trigonelline does not influence the rate that cells become labeled with [ 3 H ] - T d R or the average duration of any of the cell cycle phases. Available data demonstrate that these compounds direct cell arrest during normal cell maturation and do not suppress mitotic activity. These results show that several natural cytokinins interact with trigonelline to influence the cell cycle in two legumes. This research has demonstrated that: (1) certain natural cytokinins are antagonistic to trigonelline's promotion of cell arrest in G2 in P. sativum when present simultaneously in culture, (2) high trigonelline concentrations can abolish the effects of natural cytokinins in P. sativum, (3) certain natural cytokinins have an additive effect to trigonelline's promotion of cell arrest in G2 i n G. max when present simultaneously, (4) these natural cytokinins influence cell arrest in G2 in both legumes independently of trigonelline concentrations in roots, and synthetic cytokinins do not show any effect on trigonelline's promotion of cell arrest in G2. T o our knowledge, this is the first report of an interaction of two naturally occurring hormones that affect cell arrest in either G1 or G2 in complex tissues of either plants or animals.
Acknowledgements--Research supported, in part, by a contract with BASF, Aktiengesellschaft, Landw. Versuchsstation, Postfach 220, D-6703 Limburgerhof, West Germany and, in part, by grant GM-30290 from the Department of Health and Human Services,
87
National Institutes of Health, United States of America.
REFERENCES 1. EVANSL. S. (1979) Developmental aspects of roots of Pisum sativum influenced by the G2 Factor from cotyledons. Am. 07. Bot. 66~ 880-886. 2. EVANS L. S., ALMEIDAM. S., LYNN D. G. and NAKAmSmK. (1979) Chemical characterization of a hormone that promotes cell arrest in G2 in complex tissues. Science, Wash. 203, 1122-1123. 3. EVANSL. S. and TRAMONTANOW. A. (1981) "Is trigonelline a plant hormone?" Am. J. Bot. 6~ 1282-1289. 4. EVANS L. S. and TgAMONTANOW. A. (1984). Trigonelline and promotion of cell arrest in G2 of various legumes. Phytochemistry 2.3, 1837-1840. 5. EVANSL. S. and VAN'T HOFJ. (1974) IS the nuclear DNA content of mature root cells prescribed in the root meristem? Am. J. Bot. 61~ 1104-1111. 6. Fox J. E. (1969) The cytokins. Pages 85-123 m M. B. WILKmS, ed. The physiology of plant growth and development. McGraw-Hill, New York. 7. GODAVAgXH. R. and WaYooon E. R. (1970) Nicotinamide adenine dinucleotide metabolism in plants. I. Intermediates of biosynthesis in wheat leaves and the effect of benzimidazole. Can. J. Bot. 'l~, 2267-2278. 8. JABLONSKIJ. R. and SKOOOF. (1954) Cell enlargement and cell division in excised tobacco pith tissue. Physiol. Plant 7, 16-24. 9. LYNND. G., LEWISD. H., TRAMONTANOW. A. and EVANSL. S. (1984) Induction of cell arrest in G2: structural specificity of trigonelline. Phytochemistry 23, 1225-1228. 10. LYNN D. G., NAKANISHI K., PATT S. L., OCCOLOWlTZJ.L., ALMEIDAM. S. and EVANSL. S. (1978) Isolation and characterization of the first mitotic cycle hormone which regulates cell proliferation. 07. Am. Chem. Soc. 100, 7759. 11. MIKSlC J. R. and BROWN P. R. (1977) Complementary use of the reversed-phase and anion exchange modes of high-pressure liquid chromatography for studies of reduced nicotinamide adenine dinucleotide. J. Chromat. 142~ 641-649. 12. MILLERC. O., OKUMUgnF. S., VON SALTRAM. H. and STRONOF. M. (1956) Isolation, structure, and synthesis of kinetin, a substance promoting cell division. J. Am. Chem. Soc. 78, 1375-1380. 13. TRAMONTANO W. A., HARTNETT C. M., LYNN D. G. and EVANS L. S. (1982) Relationship between trigonelline concentration and promotion of
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cell arrest in G2 in cultured roots ofPisum sativum. Phytochemistry 21, 1201 1206. 14. TRAMONXANOW. A., LYNND. G. and EVANSL. S. (1983) Trigonelline, nicotinic acid and nicotinamide in seedlings ofPisum sativum. Phytochemistry 22, 673-676. 15. VAN'T HOFJ. and KOVACSC. J. (1972) Mitotic
cycle regulation in the meristem of cultured roots: the principal control point hypothesis. Pages 13-33 in M. W. MILLER and C. C. KUEHNERT,eds. The dynamics of meristem cell populations. Plenum Press, New York. 16. YEOMANM. M. (ed.) (1976) Cell division in higher plants. Academic Press, New York.
0098~472/85 $3.00 + 0.00 ~ 1985 Pergamon Press Ltd.
Printed in Great Britain.
EFFECTS OF C Y T O K I N I N S O N P R O M O T I O N OF CELL A R R E S T IN G2 BY T R I G O N E L L I N E A N D T R I G O N E L L I N E C O N C E N T R A T I O N S IN C U L T U R E D R O O T S OF PISUM SATIVUM A N D GLYCINE M A X A. T R A M O N T A N O , LANCE 8. EVANS a n d PAULA A. M C G I N I ~ Y Laboratory of Plant Morphogenesis, Manhattan College, The Bronx, NY 10471, U.S.A.
(Received 3 July 1984; accepted in revisedform 20 August 1984) TRAMONTANOW. A., EVANSL. S. and McGINLEY P. A. Effects ofcytokinins onpromotion of cell arrest by trigonelline and trigonelline concentrations in cultured roots of Pisum sativum and Glycine max. ENVmONMENTAL ,',NO EXPERXMENTAL BOTANY 25~ 83--88, 1985.--Effects of cytokinins on trigonelline's promotion of cell arrest in G2 were determined. Previous results demonstrate that trigonelline, (N-methyl nicotinic acid), a hormone present in cotyledons of young seedlings of Pisum sativum L. and Glycine max Merrill, is transported to roots and shoots where it stops cell progression in the G2 stage of the cell cycle during normal cell maturation. In P. sativum, two natural cytokinins, isopentenyl adenine and zeatin, and both their ribosides (all at 10-7 M) were antagonistic to trigonelline's function when placed individually in aseptic culture with trigonelline at 10- 6 M. Elevated trigonelline concentrations ( 10- 4 M) negated the antagonistic effect on these cytokinins. Kinetin and benzyl adenine (synthetic cytokinins) were not antagonistic to trigonelline at any concentration. In G. max, two natural cytokinins, isopentenyl adenine and zeatin, at 10 -7 and 10 -8 M, promoted cell arrest in G2 in the absence of trigonelline. Moreover, the proportion of cells arrested in G2 was higher in roots cultured with isopentenyl adenine in combination with trigonelline, than with either hormone alone. In both legumes, these natural cytokinins did not influence trigonelline concentrations in roots. These results demonstrate an interaction between natural cytokinins and trigonelline in control of the cell cycle in peas and soybeans. This is the first report of an interaction of two naturally occurring hormones that affect cell arrest in either G1 or G2 in tissues of either plants or animals.
INTRODUCTION
tents (in G1 a n d G2, respectively) in s t a t i o n a r y phase meristems are similar to p r o p o r t i o n s in m o r e m a t u r e root segments. ¢5) Few cells arrest d u r i n g D N A synthesis a n d no cells arrest d u r i n g mitosis. ¢5) U n d e r t e m p o r a r y c a r b o h y d r a t e deprivation, root cells o f P . sativum arrest in G 1, in G2, or become p o l y p l o i d J 1n) I f trigonelline is present, as m u c h as 6 0 % of the meristem cell p o p u l a t i o n arrests in G 2 J 1) I f sufficient trigonelline is not present, these cells arrest only in G1. ~1'1°'14) O t h e r n a t u r a l l y occurring p l a n t h o r m o n e s such as zeatin a n d isopentenyl a d e n i n e a n d their ribosides belong to a g r o u p of p h y t o h o r m o n e s called cytokinins. T h e first observed effect of cytokinins as a p l a n t h o r m o n e was the stimulation of cell division in tobacco pith callus. ~s'12) Even
TRIGONELLINE functions as a p l a n t h o r m o n e in Pisum sativum. ¢2"3'1°) Trigonelline, found in cotyledons of d r y seeds, ¢4) is t r a n s p o r t e d from cotyledons to other p l a n t tissues d u r i n g early seedling d e v e l o p m e n t ~13) a n d promotes preferential cell arrest in G2 of the cell cycle. ~2'4) Arrest of meristematic cells in the cell cycle in cultured p r i m a r y root tips after t e m p o r a r y c a r b o h y d r a t e d e p r i v a t i o n (to p r o d u c e s t a t i o n a r y phase meristems) is a n o n - r a n d o m process. Cx) Cell arrest d u r i n g n o r m a l cell differentiation (e.g. in m o r e m a t u r e segments) also occurs as a n o n - r a n d o m process. ~) Results have also shown t h a t the proportions of cells with 2C a n d 4C D N A con83
84
W.A. TRAMONTANO et al.
today, cell division activity is the most frequently observed result of cytokinins. ~6'16) Research on the control of cell division by cytokinins has occurred with individual cells and callus in aseptic cultureJ 16) The results described below point to a mechanism for control of cell proliferation in a complex tissue. The mechanism postulated herein is an interaction of cytokinins with trigonelline. Two experimental procedures were performed to determine the interactive effects of trigonelline and cytokinins. Experiments were performed to determine if cytokinins influence cell arrest in G2 alone and in combination with an effective trigonelline concentration. Since four natural cytokinins were shown herein to affect trigonelline's function, experiments were performed to determine if one of these four cytokinins (isopentenyl adenine) affected trigonelline concentrations in excised roots.
MATERIALS AND M E T H O D S
Bioassayfor promotion of cell arrest in G2 The general methods used in the bioassay have been described elsewhere, m Seeds ofPisum sativum L. and Glycine max Merrill were surface sterilized with undiluted Clorox® for 15 min, washed with sterile water to remove bleach, germinated in sterile Vermiculite®, and grown for 3 days. Under sterile conditions, excised root tips were placed in White's medium with sucrose and with various concentrations of cytokinins (Sigma Chemical Co., St. Louis, MO), with and without trigonelline. Appropriate medium controls were also used. Initially, six cytokinins at three different concentrations (10 -6, 10 .7 and 10 .8 M) were tested either with or without 10 -6 M trigonelline. Of the cytokinins tested, four were natural--isopentenyl adenine (IPA), isopentenyl adenine riboside, zeatin, and zeatin riboside. Two synthetic cytokinins, kinetin and benzyl adenine were also tested. After 3 days, root meristems were transferred to White's medium without carbohydrate or any of the test chemicals for 4 days so that cells were present only in the G1 and G2 stages of the cell cycle. The procedure used for soybeans was identical, except that seedlings were grown for 4 days before excision and the cyto-
kinins tested were IPA and zeatin at concentrations of l 0 - 7 and 10- s M. After carbohydrate deprivation, roots were fixed in a mixture of ethanol and acetic acid (3 : 1 by volume). Relative amounts of DNA per nucleus were determined on Feulgen stained nuclei from 0 to 2 mm terminal meristems by microfluorimetry. Measurements of nuclei were normalized with readings of one-half telophase and prophase figures taken to be 2C (G 1) and 4C (G2) values, respectively. (1)
Trigonelline concentrations To determine effects of cytokinins on trigonelline concentrations in cultured roots ofP. sativum and G. max, seeds were surface sterilized, washed with sterile water, germinated in sterile Vermiculite®, and grown for 3 and 4 days, respectively. Under sterile conditions, excised root tips were placed in medium with sucrose and IPA at concentrations of 10-v and 10 .8 M, with and without 10 .6 M trigonelline. Appropriate medium controls were used. After 3 days, roots were harvested and 0 1 cm terminal root segments were excised and extracted in an ethanol series. ~7,14~ After three extractions with chloroform to remove lipids, each ethanol extract was concentrated, spotted in Analtech silica gel thinlayer chromatography u.v. plates, 250 ~tm in thickness (Anahech Corp., Newark, DE 19711), and developed in acetone:water (50:50, v/v). Plates were allowed to air dry and trigonelline was eluted. Trigonelline concentrations were determined using high-pressure liquid chromatography. ~11'14) A Dupont Liquid Chromatograph Model 830 with an Ultraviolet (254 nm) detector and a Permaphase C-18 ODS column was used. For HPLC, methanol : water (50 : 50) was used as a solvent at a flow rate of 1 ml/min. ~14~ RESULTS Effects of added cytokinins on promotion of cell arrest in cultured roots ofP. sativum are shown in Table 1. The normal range for stationary phase meristems ofP. sativum is between 0.1 and 0.2 for cells in G2J 1) Cytokinins had no significant influence on the proportion of cells arrested in G2 in the absence of trigonelline. Isopentenyl adenine, trans-zeatin, and their ribosides, separately
EFFECTS OF C Y T O K I N I N S IN ROOTS OF P. S A T I V U M
85
Table 1. Antagonistic effects of natural cytokinins on promotion of cell arrest in G2 in excised roots of Pisum sativum
Concentration of cytokinin (M) 10 .7 M
10-aM
Isopentenyl adenine Isopentenyl adenine riboside Zeatin Zeatin riboside Kinetin Benzyl adenine Isopentenyl adenine Isopentenyl adenine riboside Zeatin Zeatin riboside Kinetin Benzyl adenine
None
Proportion of cells in G2 Trigonelline concentration (M) 0 10 -6 M 10 -4 M 0.17±0.05" 0.16+_0.03 0.14+_0.03 0.20+-0.02 0.17 ± 0.04 0.11 -t-0.03 0.11 +_0.01 0.13+0.03 0.11+_0.02 0.13+-0.04 0.13+-0.03 0.10+-0.02 0.10-t-0.02
0.17__.0.02 0.13±0.02 0.13+0.03 0.18__.0.03 0.38 +_0.04 0.31 +_0.04 0.30±0.06 0.14+-0.04 0.33+0.04 0.35_+0.01 0.34-t-0.02 0.32+-0.02 0.32+_0.02
0.23-t-0.03 0.39+0.03 0.34+0.03 0.31 -t-0.02 -------
* Mean+S.E. of 3 slides, 50 nuclei were scored on each slide. Each treatment was repeated three times.
at a c o n c e n t r a t i o n of 10-7 M, were antagonistic to trigonelline (10 - 6 M). I n contrast to the response of these n a t u r a l cytokinins, two synthetic cytokinins, kinetin a n d benzyl adenine, were not antagonistic to trigonelline at a n y c o n c e n t r a t i o n tested. A high trigonelline c o n c e n t r a t i o n (10 - 4 M) nullified the antagonistic effects of these n a t u r a l cytokinins at 10 - 7 M (except isopentenyl adenine). M o r e o v e r , no n a t u r a l cytokinin tested (10 - s M), except isopentenyl adenine riboside, was antagonistic to trigonelline (10 - 6 M). I n general, elevated concentrations o f trigonelline n e g a t e d the antagonistic effects of these n a t u r a l cytokinins. Cytokinins at a c o n c e n t r a t i o n of 10-6 M were not tested because they inhibited root growth, with or w i t h o u t trigonelline. These resuits d e m o n s t r a t e that the relative concentrations of trigonelline a n d n a t u r a l cytokinins in aseptic m e d i a m a y d e t e r m i n e w h e t h e r p r o m o t i o n of cell arrest in G2 in P. sativum occurs. Trigonelline concentrations within cultured roots o f P. sativum are shown in T a b l e 2. A n effective c o n c e n t r a t i o n of trigonelline was present in roots exposed to 10 - 6 M trigonelline alone (57.8 #g/g tissue). Those exposed to 10 - 6 M trigonelline with I P A (10 - v or 10 - 8 M; 52.1 and
55.2 #g/g tissue, respectively). I n contrast, when roots were exposed to 10 - 4 M trigonelline a n d 10 -T M I P A , the trigonelline concentration in roots was 390.2 #g/g tissue, far above the effective concentration for p r o m o t i n g cell arrest in G2. These d a t a suggest that the antagonistic effects
Table 2. Concentrations of trigonelline (#g/g tissue) in excised roots of garden peas (Pisum sativum) cultured for 3 days in medium with various constituents added
Treatment Control Trigonelline ( 10- 6 M) Isopentenyl adenine (10- v M) Isopentenyl adenine (10- 7 M) and trigonelline (10 -6 M) Isopentenyl adenine (10 .8 M) Isopentenyl adenine ( 10 -8 M) and trigonelline (10 -6 M)
Trigonelline cone, (/.tg/g tissue) 25.4±6.5* 57.8±6.9 37.6±3.9 52.1 +_ 10.0 29.9+ 1,0 55.2+ 10.2
* Mean+S.E. of 8 experiments in which separate sets of roots were extracted.
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W. A. TRAMONTANO et al. Table 3. Effects of natural cytokinins on promotion of cell arrest in G2 in excised roots of
Glycine max Concentration of cytokinin (M) 10-7 M 10-8 M
Isopentenyl adenine Zeatin Isopentenyl adenine Zeatin
None
Proportion of cells in G2 Trigonelline concentration (M) 0 10-6 (M) 0.35+0.02* 0.42 _ 0.05 0.36-t-0.03 0.42 +_0.04 0.22 4- 0.04
0.534-0.02 0.44 4- 0.04 0.49___0.01 0.40 4- 0.04 0.42 _ 0.02
*Mean+S.E. of 3 slides, 50 nuclei were scored on each slide. Each treatment was repeated three times.
of IPA cannot be accounted for by low concentrations of trigonelline within roots of P. sativum. Since synthetic cytokinins had no effect on trigonelline's function in P. sativum, only natural eytokinins were tested in G. max (Table 3). Both zeatin and IPA at 10 -7 and 10 - s M promoted cell arrest in G2 in the absence of trigonelline. Isopentenyl adenine in combination with trigonelline (10 -6 M) resulted in a greater proportion of cells arrested in G2 compared with only one compound present. These results demonstrate that IPA has an additive effect for promoting cell arrest in G2 by trigonelline. Zeatin in combination with trigonelline did not produce this additive effect. Trigonelline concentrations in cultured roots of G. max, were higher than controls when cultured with 10 -6 M trigonelline (Table 4). IPA did Table 4. Concentrationsof trigonelline (#g/g tissue) in excised roots of soybeans (Glycine max) culturedfor 3 days in medium with various constituents added
Trigonelline conc.
Treatment No additives Isopentenyl adenine (10-s M) Trigonelline ( 10- 8 M) Isopentenyl adenine (10-s M) and trigonelline (10 -6 M)
(pg/g tissue) 12.04-2.4" 17.14-6.1 42.14-8.2 35.94-1.0
* Mean 4-S.E. of results of 4 experiments in which separate sets of roots were extracted.
not affect trigonelline concentrations in cultured roots of G. max under any conditions. These results suggest that IPA promotes cell arrest in G2 in G. max independent of root trigonelline concentrations.
DISCUSSION Natural cytokinins affect trigonelline's function both in P. sativum and G. max. In P. sativum, cytokinins have an antagonistic effect on trigonelline's promotion of cell arrest in G2. This antagonism by cytokinins can be overridden by relatively high trigonelline concentrations. In this manner, promotion of cell arrest in G2 may be dependent on relative concentrations of trigonelline and cytokinins. The antagonistic effects of natural cytokinins on trigonelline's function in P. sativum cannot be explained by changes in trigonelline concentrations within roots. These results suggest that the antagonistic effects of cytokinins in P. sativum cannot be explained by regulation of trigonelline metabolism within the pyridine nucleotide pathway. In contrast to the situation with peas, natural cytokinins promoted cell arrest in G2 in G. max. In general, the effects of isopentenyl adenine were additive to those of trigonelline in this species. From the available data it seems that isopentenyl adenine promoted cell arrest in G2 independently of trigonelline in G. max. A difference in function between synthetic and natural cytokinins is demonstrated. Although no
EFFECTS OF CYTOKININS IN ROOTS OF P. S A T I V U M explanation is presently available for this difference, it is possible that the structural specificity ofcytokinins governs the inhibition oftrigonelline function in peas in the same way that the structural specificity of trigonelline is a requirement for promotion of cell arrest in G2. (9) Isopentenyl adenine and zeatin are alike structurally, the only difference between the two compounds is a hydrogen in I P A which is substituted by a hydroxyl group in zeatin. Moreover, zeatin and I P A are similar in function in relation to effects of trigonelline function. Kinetin and benzyl adenine are not similar in structure and function to the two natural cytokinins. It m a y be questioned whether trigonelline and/or natural cytokinins suppress the cell cycle or change the average phase durations. From available evidence (1, unpublished results) it seems that trigonelline does not influence the rate that cells become labeled with [ 3 H ] - T d R or the average duration of any of the cell cycle phases. Available data demonstrate that these compounds direct cell arrest during normal cell maturation and do not suppress mitotic activity. These results show that several natural cytokinins interact with trigonelline to influence the cell cycle in two legumes. This research has demonstrated that: (1) certain natural cytokinins are antagonistic to trigonelline's promotion of cell arrest in G2 in P. sativum when present simultaneously in culture, (2) high trigonelline concentrations can abolish the effects of natural cytokinins in P. sativum, (3) certain natural cytokinins have an additive effect to trigonelline's promotion of cell arrest in G2 i n G. max when present simultaneously, (4) these natural cytokinins influence cell arrest in G2 in both legumes independently of trigonelline concentrations in roots, and synthetic cytokinins do not show any effect on trigonelline's promotion of cell arrest in G2. T o our knowledge, this is the first report of an interaction of two naturally occurring hormones that affect cell arrest in either G1 or G2 in complex tissues of either plants or animals.
Acknowledgements--Research supported, in part, by a contract with BASF, Aktiengesellschaft, Landw. Versuchsstation, Postfach 220, D-6703 Limburgerhof, West Germany and, in part, by grant GM-30290 from the Department of Health and Human Services,
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National Institutes of Health, United States of America.
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