The effect of inhibitors and promoters of poly(ADP-ribosyl)ation on cell arrest in G2 caused by trigonelline in roots of Pisum sativum

Environmental and Experimental Botany, Vol. 27, No. 4, pp. 4 6 3 4 7 2 , 1987 Printed in Greal Britain.

01198-8472/87 $3.00 + 0.00 ~: 1987. Pergamon Journals Ltd.

THE EFFECT OF INHIBITORS AND PROMOTERS OF POLY(ADPRIBOSYL)ATION ON CELL ARREST IN G2 CAUSED BY TRIGONELLINE IN ROOTS OF PISUM S A T I V U M WILLIAM A. T R A M O N T A N O , FRANCENE M. GALLOUSIS and DONNA A. PHILLIPS

Laboratory of Plant Morphogenesis, Manhattan College, Bronx, NY 10471, U.S.A.

(Received 19 December 1986; accepted in revisedform 10 April 1987) TRAMONTANOW. A., GALLOUSISF. M. and PHILLIPSD. A. The ~'ect of inhibitors and promoters of poly( ADP-ribosyl)ation on cell arrest in G2 caused by trigoneUine in roots of Pisum sativum, ENVIRONMENTAL AND EXPERIMENTALBOTANY 27, 463 472, 1987. Experiments were performed to determine if compounds which are known to affect poly(ADP-ribosyl)ation could alter the promotion of G2 arrest caused by trigonelline in aseptic culture. Several known inhibitors of poly(ADP-ribose) formation, such as 7-methylxanthine and 3-methoxybenzamide, when added to White's culture media at 10 -4 M were antagonistic to trigonelline. Other known inhibitors of poly(ADP-ribose) formation had no effect on trigonelline's G2 promoting ability. Several polyamines, which possess poly(ADP-ribose) promoting ability, had no influence on trigonelline's function. 7-Methylxanthine and 3-methoxybenzamide did not affect the labeling or mitotic indices of meristems. However, these two compounds altered the metabolism of ~4C-carbonyl NAD. Both compounds diminished the uptake of ~4C-carbonyl NAD and both decreased the rate of synthesis of trigonelline. With 3-methoxybenzamide, most of the radiolabel in the media was nicotinamide after 48 hr. From the data reported herein, since several of the poly(ADPribose) synthesis inhibitors appear to antagonize trigonelline and alter the metabolism of NAD, the mechanism of G2 arrest caused by trigonelline is postulated to be mediated via poly(ADPribose). INTRODUCTION

naturally occurring cytokinins, as known plant cell division stimulators, block trigonelline's funcIx has been 8 years since the isolation and chartion in cultured pea rootsJ 25~ acterization of the G2 factor, trigonelline (NAt the present time, no mechanism has been methyl nicotinic acid), from the cotyledons of postulated by which trigonelline promotes cell garden peas (Pisum sativum L.)./5/ Trigonelline arrest in G2. Evidence from both animal and promotes cell arrest in the G2 phase of the cell plant cells suggests that poly(ADP-ribose) is cycle in excised roots of several legumes./6/ Triinvolved in the regulation of cell proliferation and gonelline, a methylated derivative of nicotinic progression through the cell cycle. (12'24'33i Since acid, is a m e m b e r of the pyridine nucleotide pathtrigonelline is a cell cycle regulator in peas, and way which produces nicotinamide adenine di- is also a member of the pyridine nucleotide pathnucleotide.i~9/Exposure to other members of this way, the formation of poly(ADP-ribose) m a y be pathway in excised roots results in a rapid con- involved in the G2 promoting ability of trigonelversion to trigonelline, thereby increasing the per- line. Poly(ADP-ribose) is formed essentially via a centage of cells in G2./27/ Once trigonelline has two step process: first, N A D is cleaved to yield been synthesized, it is quite stable, and shows nicotinamide (NAM) and ADP-ribose, next, the little demethylation to nicotinic acid./27/ Several ADP-ribose units are polymerized by the action 463

464

W . A . TRAMONTANO

of poly(ADP-ribose) synthetase./17~ Much of the recent research with poly(ADP-ribose) has utilized the presence or absence of nicotinamide and nicotinic acid. These substances may act as inhibitors ofpoly(ADP-ribose) synthesis. !2)Both of these compounds are closely linked with trigonelline in the pyridine nucleotide cycle. Previous experiments have shown that N A M levels in the root are low ( < 1 #g/g), and i f l x l 0 4 M NAMis given to excised roots in White's medium, most of this becomes trigonelline, c26'271I f l x 10 4 M NAD is given to excised roots in White's medium, a majority of the NAD is converted to trigonelline,/26i possibly leading to the formation of poly(ADP-ribose) from the remaining ADP-ribose units. It appears that there exist some interconnections among trigonelline, cell cycle regulation, poly(ADP-ribose) and the pyridine nucleotide cycle. Poly(ADP-ribosyl)ation is a post-translational protein modification which, like histone acetylation, appears to be correlated with regulatory processes controlling DNA-related activities of the nucleus, such as DNA synthesis, '8/sister-chromatid exchange, (15/ and DNA repair. (3'16/ In mammalian cells, massive DNA strand breakage induced by alkyl-nitroso compounds triggers a severe drop in NAD levels./1°/ The most common method to study the role of (ADP-ribosyl)ation in cell function is to use inhibitors of the chromatin-bound enzyme poly(ADP-ribose) synthetase, which is essential for poly(ADP-ribose) formation. Both methylated xanthines and cytokinins have been shown to be effective inhibitors of ribosylation] ~3/ As stated previously, natural cytokinins antagonize trigonelline's G2 arrest in peas. Many laboratories have investigated the possible involvement of (ADP-ribosyl) ation of nuclear proteins in relation to regulatory events of the cell cycle. Although no consistent theory or concept has emerged, some results suggest that the synthesis of poly(ADP-ribose) is lowest during Sphase and highest in either G1 or G2. (8/In intact and disrupted nuclei of H e L a $3 cells the highest amount of poly(ADP-ribose) synthesis was demonstrated in the G2 phase./24/A 6-fold increase in poly(ADP-ribose) during S phase, followed by a 10-fold increase during the G2 phase, was also shown in H e L a cells./12)

et al.

H e L a cells were also incubated with dimethyl sulfate in the presence or absence of the poly(ADP-ribose) synthesis inhibitor benzamide. The cell cycle distribution was analyzed after 24 hr and an accumulation of cells in G2 occurred when benzamide was present] 3tl The presence of 3aminobenzamide (3-AB) following X-ray irradiation accumulated cells in G2. c2~i 3Methoxybenzamide (3-MB) at 1 m M inhibited the formation of poly(ADP-ribose) in a cultured cell line./9/ When Chinese hamster ovary cells were exposed to 3-AB, a delay of entry of cells into S was shown."2°) 3-AB when given to cultured human fibroblasts with DNA damage interfered with the DNA repair process, causing cells to arrest in G2. (l/ This information suggests that ADP(ribosyl)ation reactions may be involved with cell cycle regulation, since inhibitors ofpoly(ADP-ribose) formation display such varied effects on the cell cycle. The evidence for poly(ADP-ribosyl)ation in plants is certainly far less than that for animal cells. Thirty-three percent of onion root tip nuclei displayed intense incorporation of 3H-NAD after a brief exposure to the isotope./;a/ The incorporation of 14C NAD into nuclei isolated from cultured tobacco cells was also demonstrated./32:~ Inhibitors of ADP-ribose formation such as 3AB prevented the incorporation of 14C NAD into wheat embryo nuclei./3°/ With wheat embryos a 2-4 fold stimulation in the rate of poly(ADPribose) synthesis was found when polyamines were present. 129) Some evidence has shown that several polyamines regulate the cell cycle in plants./~z/ Moreover, several studies have shown that poly(ADP-ribosyl)ation is stimulated by the polyamine spermine. (22,23) Root meristems of legumes in particular are excellent model systems because of their: (a) ease of accessibility, (b) ability to respond to compounds in aseptic growth media, (c) ease of tritiated thymidine labeling for cell kinetic studies, and (d) for garden pea nuclei, relatively large amount of nuclear DNA. Several years ago it was proposed that poly(ADP-ribosyl)ation in the plant nucleus may be influenced by the action of phytohormones./33/ Since trigonelline controls the proportion of cells present in the G2 phase of the cell cycle, and appears to work with the cytokinins in controlling

INHIBITORS AND PROMOTERS OF PISUM SAT1VUM G2 arrest, the possible link(s) among these factors was investigated. Experiments were performed to determine if (a) inhibitors and promoters ofpoly(ADP-ribose) synthesis affected the ability oftrigonelline to promote cell arrest in G2, (b) some of the inhibitors tested affected the labeling and mitotic indices of meristematic nuclei, and (c) the rate and extent of labeling of 14C-carbonyl NAD into other members of the pyridine nucleotide pathway under the influence of several inhibitors of poly(ADPribose) synthesis are affected. MATERIALS

AND METHODS

1. General culture conditions Seeds of Pisum sativum L. (garden peas) cv. Alaska 2B were surface sterilized with undiluted Clorox '~' that contained 5.25% sodium hypochlorite, stirred frequently for 10 min, washed with sterile water to remove bleach, and germinated in the dark at 23°C in sterile vermiculite.

465

sent in White's medium with sucrose before establishment of stationary phase, then a larger proportion ( ~ 0.40-0.60) of cells arrested in G2.17/

3. D NA measurements Measurements by relative DNA per nucleus of Feulgen-stained nuclei were obtained by microfluorimetry. The incident light passed through a 545 nm interference filter (9 nm band width) and was focused with a dark field condenser onto individual Feulgen-stained nuclei. Feulgen stain fluorescence of light greater than 580 nm was recorded by a red-sensitive photomultiplier tube (Microspectrum Analyzer, Model 138119, Farrand Corp. Valhalla, NY). The relative amount of DNA per nucleus was normalized with readings ofone-halftelophase and prophase mitotic figures taken to be 2C and 4C values, respectively. Cells with or near a 2C DNA value were assumed to be in G1, while those with a 4C DNA value were assumed to be in G2.

4. aH- TdR labeling of meristem cells

Experiments were performed to determine the rate that cells became labeled with 3H-TdR as a 2. Bioassa~J'orpromotion of cell arrest in G2 General culture techniques to determine the measure of the proliferative capacity of the merability of trigonelline to promote cell arrest istem population. If the rate of labeling differed in G2 by a standardized bioassay have been among treatments it would indicate a change in described. ~4~ Under aseptic conditions excised the number of proliferative cells, the cell cycle root meristems from 3-day-old seedlings were duration, or some other perturbation. Freshly placed in White's medium with sucrose for 3 days excised 3-day-old roots were cultured in White's medium with sucrose and tritiated thvmidine betbre transfer to White's medium without sucrose for 3 days. This period of carbohydrate (185 mBq/ml). Tritiated thymidine :~H-TdR was deprivation establishes a stationary phase meri- present in culture conditions with either 1 x 10 " stem. A stationary phase meristem is defined as a M trigonelline, a poly ADP-ribose inhibitor or meristem (0-2 mm portion) in which progression both. Samples were taken after 45 min, 8 hr and through the cell cycle has ceased temporarily. 17~ 16 hr. Roots were fixed (3:1, 95~}~ EtOH:glacial In some experiments trigonelline (Sigma Chemi- acetic acid), stained with Feulgen, and the tercal Co., St Louis, MO) and/or a poly(ADP- minal 0-2 mm meristem was squashed on a slide ribose) inhibitor or promoter was added to 50 ml and dipped in Kodak NTB-1 emulsion (Kodak of White's medium with sucrose which normally Corp., Rochester, NY 14650). After 2 3 weeks supports growth of 10 excised roots. With event- exposure, slides were developed, and prepared for ual establishment of a stationary phase by tem- microscopy. Labeled nuclei possessed more than porary carbohydrate deprivation, cells were five silver grains per nucleus while nuclei with five arrested in G1 and in G2 (2C and 4C contents, grains or fewer were considered unlabeled. This respectively) within the terminal meristem. If established criterion is employed to prevent overroots ti-om 3-day-old seedlings were placed in estimating the numbers of labeled cells. White's medium without any additives before 5. Incorporation of ~4C-NAD into other memberr of the establishment of stationary phase, only 0.20 cells pyridine nucleotidepathway arrested in G2. However, if a sufficient conTo determine if an inhibitor of poly(ADPcentration of trigonelline (1 x 10 6 M) was pre- ribose) synthesis affected the rate and extent of

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W . A . T R A M O N T A N O et al.

14C-carbonyl N A D (1.85 mBq/ml) into other members of the pyridine nucleotide pathway, excised 3-day-old roots were placed into 1 x 10 -4 M 14C-carbonyl NAD, added to White's medium with sucrose either with or without either 7-methylxanthine (7-MX) or 3-MB. After 3 days in culture with these additives, roots were immersed in methanol, extracted with chloroform, and reduced to 1 ml/g of tissue. Media and extracts were plated on Analtech Silica gel G F thin-layer plates, 250 # m thickness; 200 #1 of extract was applied to each plate along with nonradioactive nicotinic acid, nicotinamide, NAD, trigonelline and nicotinamide mononucleotide to facilitate component identification. Plates were developed in both acetone:acetic acid:methanol:benzene, 1:1:4:14 v/v for localization of nicotinic acid and nicotinamide and ethanol: 1 M a m m o n i u m acetate, 5:2 v/v for localization oftrigonelline, nicotinamide mononucleotide and NAD. After development, silica that corresponded to the five components above and areas between these known components was scraped from the plates. Silica from known components and the areas between components were placed in individual liquid scintillation vials with Aquasol. '~' All vials were counted in a Beckman LS-150 Scintillation Counter. The a m o u n t of radioactivity in each component was calculated from quench curves and corrections of specific activity. Since most of the radioactivity was associated with these components only estimates of activities of these constituents were reported. The total a m o u n t of radioactivity on each plate was determined by adding values from all plate areas. Aliquots of root extracts and media were counted directly so that all values could be adjusted to determine the concentration of these components in both roots and media, respectively.

RESULTS

T h e first series of experiments was performed to determine whether inhibitors or possible promoters ofpoly(ADP-ribosyl)ation could promote cell arrest in G2 when placed in the bioassay alone, or if placed simultaneously with trigonelline, could they inhibit the G2 arresting ability of

trigonelline. D a t a presented in Table 1 show that after 3 days exposure to the test c o m p o u n d prior to the establishment of stationary phase, of all the xanthine derived compounds, only hypoxanthine at both l x 1 0 _4 M and l x l 0 6 M, and 7M X at l x l 0 4 M were able to block the G2 promoting ability of trigonelline. No c o m p o u n d alone promoted cell arrest in G2 after this exposure period. Hypoxanthine is not a documented inhibitor of poly(ADP-ribose), but it is commonly found in garden peas. 7 - M X is a welldocumented inhibitor of poly(ADP-ribose) synthesis. Data presented in Table 2 show that several of the benzamide derived compounds tested were able to antagonize trigonelline's G2 promoting ability when exposed to the same procedures as described in the preceding paragraph. None of these compounds alone promoted cell arrest in G2. Both 3-MB and 3-nitrobenzamide (3-NB) at 1 x 10 -4 M were effective in antagonizing trigonelline. Both of these compounds are known inhibitors of poly(ADP-ribose) synthesis. The most well-documented inhibitor of poly(ADPribose) synthesis, 3-AB, did not inhibit trigonelline's G2 promoting ability. W h e n polyamines were placed alone in the bioassay (Table 3), no promotion of G2 arrest was demonstrated. No blockage or enhancement of G2 occurred when added simultaneously with trigonelline. The results of the above experiments suggested that some inhibitors of poly(ADP-ribose) synthesis were able to antagonize trigonelline. Next, these inhibitors of trigonelline function were tested to determine if they altered the rate that meristem cells incorporate SH-TdR, after exposure times of 45 min, 8 hr and 16 hr. W h e n 3-day-old roots were placed into White's culture medium with sucrose, the mitotic index decreased to near zero by 8 hr, but as the roots acclimated to the new conditions, cell progression through the cell cycle approached normal rates. '2~~At 45 min about 15 20% of the nuclei were labeled in the majority of treatments (Table 4). Only 2 treatments, 3-NB 1 x 10 -4 M and 3-MB 1 x 10 .-4 M, exhibited a labeling index of about 60r~/o of controls. At the 8 hr exposure time, these treatments along with 3-MB 1 x 10 -4 M, trigonelline 1 x 10 -6 M had lower labeling indices, being

INHIBITORS

AND PROMOTERS

OF PISUM SATIVUM

Table 1. Effects of xanthine and its derivatives on the promotion of cell arrest in G2 by trigonelline in stationary phase meristems of garden peas exposed to the test compoundfor 3 days

C o n c e n t r a t i o n of inhibitor (M) None X a n t h i n e (10 ~ M) H y p o x a n t h i n e ( 10 - 4 M) H y p o x a n t h i n e ( 10 -~ M) 3 - M e t h y l x a n t h i n e ( 10 4 M) 7 - M e t h y l x a n t h i n e (10 ~ M) 7 - M e t h y l x a n t h i n e (10 6 M) Caffeine (10 4 M) ( 1,3,7-trimethylxanthine) Theophylline ( 10 - 4 M) ( 1,3-dimethylxanthine) T h e o b r o m i n e (10 4 M) (3,7-dimethylxanthine) T h e o b r o m i n e ( 10 -6 M)

Proportion of cells in G2 Trigonelline concentration (M) 0 10 6 M 0.15-t-0.01" 0.16-t-0.03 0.17 + 0.02 0.21 ___0.02 0.17 -I- 0.04 0.17___0.01 0.16-t-0.02

0.33___0.02]" 0.37+__0.01 0.21 _+0.02 0.23 -I- 0.02 0.33 + 0.01 0.17±0.03 0.34±0.02

0.13 ± 0.02

0.36 ___0.02

0.14 + 0.02

0.35 + 0.03

0.15 4- 0.02 0.15 _+0.03

0.31 _+0.02 0.32 _ 0.01

* M e a n _ S.E. of three slides, 50 nuclei were scored on each slide. Each experiment was repeated twice. ]. Student's t-test showed significant difference at the 95%0 confidencc level between the means at the top of each column.

Table 2. Effects of benzamide and its derivatives on the promotion of cell arrest in G2 by trigoneUine in stationary phase meristems of garden peas exposed to the test compound for 3 days

C o n c e n t r a t i o n of inhibitor None Benzamide (10 4 M) Benzamide (10 -6 M) 3 - M e t h o x y b e n z a m i d e ( 10-4 M) 3 - M e t h o x y b e n z a m i d e (10 6 M) 3 - A m i n o b c n z a m i d e (10-4 M) 3 - A m i n o b e n z a m i d e ( 10 -6 M) 3 -Ni trobenzamide ( 10 - 4 M) 3-Nitrobenzamide (10 6 M)

Proportion of cells in G2 Trigonelline concentration (M) 0 10 6 M 0.15+0.01" 0.16+0.01 0.15-t-0.01 0.17-t-0.03 0.14 + 0.02 0.17+0.02 0.15-1-0.02 0.13+0.02 0.14-t-0.02

0.33_+ 0.02"~ 0.33-t-0.01 0.34__.0.02 0.13+0.03 0.25 _ 0.04 0.36___0.02 0.34-t-0.03 0.14+0.03 0.32-t-0.02

* M e a n + S.E. of three slides, 50 nuclei were scored on each slide. Each experiment was repeated twice. ]. Student's t-test showed s i g n i f c a n t differences at the 95~!"o confidence level between the means at the top of each column.

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W. A. T R A M O N T A N O et al.

468

Table 3. Effects of polyamines on the promotion of cell arrest in G2 by trigonelline in stationary phase meristems of garden peas exposed to the test compoundfor 3 days

Concentration of polyamine None Spermine (10 4 M) Sperm±dine ( 10 - 4 M) Cadaverine (10 -4 M) Putrescine (10 -~ M)

Proportion of cells in G2 Trigonelline concentration (M) 0 10 6M 0.15±0.01" 0.15±0.02 0.15 _. 0.04 0.14±0.02 0.11 ±0.02

0.33 ± 0.02"~ 0.36±0.01 0.34 __0.01 0.34±0.02 0.33 ±0.03

* Mean _ S.E. of three slides, 50 nuclei were scored on each slide. Each experiment was repeated twice. "~Student's t-test showed significant differences at the 95(}/o confidence level between the means at the top of each column.

Table 4. Effects of trigonelline, and various inhibitors of poly(ADP-ribose) synthetase on the incorporation of [3H]- TdR in 0-2 mm root meristems of Pisum sativum cultured in White's medium with sucrosefor various time periods

Conc. of additive

Control Trigonelline (10 ~ M) Hypoxanthine (I0 4 M) Hypoxanthine ( 10 -.4 M) Trigonelline ( 10 - 6 M) 7-Methylxanthine (10 -4 M) 7-Methylxanthine (10-~ M) Trigonelline (10 -6 M) 3-Aminobenzamide ( 10 -4 M) 3-Aminobenzamide (10 4 M) Trigonelline (10 6 M) 3-Nitrobenzamide (10 ~ M) 3-Nitrobenzamide (10 4 M) Trigonelline (10 ~ M) 3-Methoxybenzamide (10 4 M) 3-Methoxybenzamide (10 -~ M) Trigonelline (10 -6 M)

Duration of exposure to [3H]-TdR Percent cells" which incorporated 3H - TdR 45 min 8 hr 16 hr

20.0±2.1" 44.8±2.3 16.6±2.5 40.6±2.6 17.8±0.6 45.9±3.1

63.2±0.6 55.3±5.6 57.3±2.1

18.2±1.5 20.9±1.1

39.1±1.7 43.5±3.6

60.7±3.7 59.8±1.5

18.1±2.0 15.1±2.4

41.5±1.6 37.0±0.8

55.0±2.8 55.7±1.5

19.2±1.4 10.9±0.9

40.8±1.4 36.1±1.4

58.8±2.2 55.6±2.9

15.6±2.9 11.0±0.6

42.4±2.7 32.6±1.2

55.9±3.1 54.7±1.5

14.9±2.4

34.1±1.2

57.0±2.0

* Mean and S.E. of three slides, 1000 nuclei were scored on each slide. Each treatment was performed twice.

a b o u t 6 0 - 7 0 % of controls. However, by 16 hr, all treatments had similar indices (55-60%) indicating that blockage in the cell cycle had not occurred. T h e initial lower labeling indices of

some treatments might indicate a general reduction in cell cycle activity. However, a general reduction in mitotic cycle activity was not indicated from analysis of mitotic indices of

INHIBITORS AND PROMOTERS OF P I S U M S A T I V U M

469

Table 5. Effects of trigonelline and various inhibitors of poly( AD P-ribose ) smthetase on the mitotic index ofO 2 mm root meristems ofPisum sativum, exposed to [3H]- TdR for 45 min in White' s medium with sucrose

Conc. of additive

Percent cells in mitosis

Control Trigonelline ( 10 - 6 M) Hypoxanthine (10 4 M)

Hypoxanthine (10 -4 M), Trigonelline (10 6 M) 7-Methylxanthine ( 10 -4 M) 7-Methylxanthine (10 4 M), Trigonelline (10 GM) 3-Aminobenzamide (10 4 M) 3-Aminobenzamide ( 10 4 M), Trigonelline ( 10- ~sM) 3-Nitrobenzamide (10 4 M) 3-Nitrobenzamide ( 10 - ~ M), Trigonelline ( 10 ~ M) 3-Methoxybenzamide (10 4 M) 3-Methoxybenzamide (10 4 M), Trigonelline (10 ~ M)

4.4±0.6* 5.1±0.5 4.6±0.8 4.9±O.2 5.8±0.3 4.6±0.4 5.5±0.1 6.5±0.1 7.1±0.9 5.2±1.1 7.0±0.6 6.0±0.6

* Mean and standard error of three slides, 1000 nuclei were scored on each slide. Each treatment was performed twice.

samples exposed to the test c o m p o u n d for 45 min in White's m e d i u m with sucrose (Table 5). I n the last series of experiments, excised 3-dayold roots were exposed to ~4C-carbonyl N A D at 1 x 10 4 M, either with or w i t h o u t 1 × 10 -4 M 7 - M X or l x l 0 4 M 3-MB for 24 or 48 h r i n White's m e d i u m with sucrose. These two compounds were chosen since each at 1 x 10 .4 M was antagonistic to trigonelline (Tables 1-2) a n d each has been shown to be an inhibitor of poly(ADPribose) formation. Extracts from these roots were prepared a n d analyzed via T L C a n d liquid scintillation counting. D a t a presented in T a b l e 6 show the percentage of the total radioactivity (combined root extracts a n d media) a t t r i b u t e d to specific pyridine nucleotide intermediates. T h e percentage of total radioactivity in root extracts was related with exposure time. For all the treatments a range of 2.2-3.6% of all radioactivity was present in the root extracts at 24 hr a n d there were no significant t r e a t m e n t effects. At 48 hr of exposure, 10.9~o of the total radioactivity added to 1 x 10 4 M N A D culture m e d i u m was present in root extracts. However, if the m e d i u m also contained 7 - M X or 3-MB, each at 1 x 10 .4 M, then the percentages of radioactivity in root extracts were 8.8 a n d 4.1 °'o, respectively. U p t a k e of labeled N A D was markedly lower in the latter situation.

T h e percent of radioactivity in the 48 hr extracts that can be attributed to trigonelline varied significantly. I n the N A D l x 10 .4 M treatment, 9.2°% of the total radioactivity, 84°~, of the radioactivity limited to the root extracts, was present as trigonelline. This value fell to 5.5% of the total radioactivity, 60°/{~of the radioactivity in the root extracts for 7 - M X 1 x 10 4 M treatment. This was further depressed in the 3-MB 1 x 10 4 M t r e a t m e n t to 2.4% of the total radioactivity, or 58% of the radioactivity present in the roots. No other significant features were present a m o n g the treatments. O f the other pyridine intermediates examined in the root extracts, several overall trends were evident. T h e overall percent radioactivity attributed to nicotinic acid, n i c o t i n a m i d e a n d N A D fluctuated little a m o n g the treatments. A n i m p o r t a n t aspect of this research was to detect the possible presence of n i c o t i n a m i d e mononucleotide. I n these experiments no n i c o t i n a m i d e mononucleotide was detected in a n y treatment. This was significant since a large a m o u n t of this c o m p o u n d might suggest an inability to form poly(ADP-ribose) because if cleavage of N A D occurred between the phosphate groups to yield n i c o t i n a m i d e mononucleotide, the core ADP-ribose moiety would not be present.

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TRAMONTANO

et al.

Table 6. Percentage of radioactivity within roots and media of various components of the pyridine nucleotide pathway when excised Pisum sativum roots were culturedfor either 24 or 48 hr in the presence of [HC-carbonyl] NAD either alone or in combination with 7-methylxanthine (10 4 M) or 3-methoxybenzamide 10 -4 M)

Root extracts* (% total)~" Components

Media* (% total)'~ NAD Trig

Total Others + in media

Total radioactivity

Treatment

(hr)

Total m NA NAM Trig NAD Others++ extracts NAM

NAD (10 4 M)

0 24 48

0.2 0.5

0.6 0.4

0.6 9.2

0.3 0.1

0.5 0.7

2.2 10.9

3.5 3.1 6.7

95.3 91.7 78.8

1.2 3.0 4.1

100 97.8 89.1

100 100 100

NAD (10 4 M) 7-MX (10 -4 M)

0 24 48

0.3 0.7

0.5 1.1

1.3 5.5

0.2 0.4

1.0 1.1

3.3 8.8

4.6 4.7 9.8

94.7 91.0 70.6

0.7 1.0 10.8

100 96.7 91.2

100 100 100

NAD (10 ~ M) 3-MB (10 -4 M)

0 24 48

0.2 0.4

1.0 0.7

1.1 2.4

0.4 0.2

0.9 0.4

3.6 4.1

11.8 17.0 34.5

86.9 77.7 57.9

1.3 1.7 3.5

100 96.4 95.9

100 100 100

'1'ime

* All calculations are corrected for 30 ml of media, which supported the growth of 10 roots. All root extracts arc g/ml and corrected for the appropriate volume. t Percentage of radioactivity in each component recovered from either media or roots was calculated on the basis of the total radioactivity in media plus roots. ++The phrase 'others' refers to all other areas of the thin-layer plates not related to the four components assayed.

E x a m i n a t i o n o f the r a d i o a c t i v i t y p r e s e n t in the m e d i a s h o w e d a g e n e r a l d e c l i n e in the a m o u n t of r a d i o a c t i v i t y p r e s e n t w i t h time, the g r e a t e s t d e c r e a s e o c c u r r i n g in the N A D at 1 x 10 -4 M roots. T h e p e r c e n t a g e o f r a d i o a c t i v i t y in the m e d i a t h a t c o u l d be assigned to n i c o t i n a m i d e rose m o d e r a t e l y w i t h t i m e in b o t h the 1 x 10 -4 M N A D a n d the l x l 0 4 M N A D , l x 1 0 .4 M 7 - M X t r e a t m e n t . H o w e v e r , a r a p i d rise in the a m o u n t o f n i c o t i n a m i d e in the m e d i a o c c u r r e d in the N A D l x l 0 -4 M , 3 - M B at l x l 0 4M t r e a t m e n t , s u g g e s t i n g t h a t the f o r m a t i o n o f nico t i n a m i d e f r o m N A D was o c c u r r i n g , b u t t h a t the n i c o t i n a m i d e was n o t r e m a i n i n g in the r o o t b u t was b e i n g released b a c k into the m e d i a . T h i s p h e n o m e n o n has b e e n d e m o n s t r a t e d p r e v i o u s l y w i t h o t h e r p y r i d i n e i n t e r m e d i a t e s J TM T h e r e was no e v i d e n c e o f n i c t o t i n a m i d e m o n o n u c l e o t i d e in the m e d i a , since o n l y b a c k g r o u n d levels o f r a d i o a c t i v i t y w e r e d e t e c t e d on T L C plates at the l o c a t i o n w h e r e the p u r e n i c o t i n a m i d e m o n o n u c l e o t i d e ran. DISCUSSION

T h e r e exists a t r e m e n d o u s g a p b e t w e e n the a m o u n t o f i n f o r m a t i o n on p o l y ( A D P - r i b o s y l )

a t i o n in a n i m a l systems vs t h a t fi)r p l a n t systems. M o s t o f o u r c u r r e n t i n f o r m a t i o n on poly( A D P - r i b o s e ) has b e e n d e r i v e d f r o m a n i m a l tissue c u l t u r e lines. W i t h the d i s c o v e r y of t r i g o n e l l i n e as a n a t u r a l l y o c c u r r i n g G2 p r o m o t e r in legumes, it w o u l d a p p e a r t h a t a system is n o w a v a i l a b l e in w h i c h one c a n r e g u l a t e the p e r c e n t o f cells in G 2 in excised roots by the p r e s e n c e or a b s e n c e o f this p h y t o h o r m o n e . P e r h a p s , as the p e r c e n t a g e s o f cells in G 2 v a r y w i t h t r i g o n e l l i n e e x p o s u r e , the levels a n d rates o f p o l y ( A D P - r i b o s y l ) a t i o n of n u c l e a r p r o t e i n s v a r y as well. B e c a u s e o f the insufficient a m o u n t o f e x p e r i m e n t a t i o n d o n e on p l a n t systems, m a n y o f the e x p e r i m e n t s r e p o r t e d h e r e i n w e r e d e r i v e d ti'om the results o f e x p e r i m e n t s p e r f o r m e d on a n i m a l tissue c u l t u r e cells. S e v e r a l o f the k n o w n poly( A D P - r i b o s e ) synthesis i n h i b i t o r s w e r e able to a n t a g o n i z e t r i g o n e l l i n e w h e n p r e s e n t at 1 x 10 4 M, h o w e v e r this effect was lost in s o m e cases w h e n the i n h i b i t o r ' s c o n c e n t r a t i o n was r e d u c e d to 1 x 10 -6 M . It w o u l d a p p e a r t h a t these inhibitors h a v e a p h y s i o l o g i c a l effect in pea root m e r i s t e m cells. W h e t h e r or not this p h y s i o l o g i c a l effect can be definitely a t t r i b u t e d to a lack or presence o f p o l y ( A D P - r i b o s e ) f o r m a t i o n r e m a i n s to be determ i n e d . 3-AB has r e c e n t l y b e e n s h o w n to h a v e a

I N H I B I T O R S AND P R O M O T E R S OF PISUM S A T I V U M wide range of effects on D N A precursor m e t a b olismJ TM This has cast some d o u b t on the specificity of 3-AB inhibition to p o l y ( A D P - r i b o s e ) synthetase. P e r h a p s the loss of the G2 effect with 1 x 10 -4 M 3-AB, 1 x 10 -6 M trigonelline is due to an i n h i b i t o r y effect on D N A precursor m e t a b olism. I t does a p p e a r t h a t none of the c o m p o u n d s tested either affected the progression of the meristem cells t h r o u g h the cell cycle or disturbed the mitotic index. F r o m these experiments, it would a p p e a r that these inhibitors do have an effect on the m e t a b olism of N A D within the p y r i d i n e nucleotide cycle. T h e u p t a k e a n d m e t a b o l i s m of N A D were altered when the inhibitors were present. I n the case of 3-MB, the a m o u n t of r a d i o a c t i v i t y in the root extract was greatly diminished, while the a m o u n t o f n i c o t i n a m i d e which was present in the m e d i a was enhanced. As stated previously, an a b u n d a n c e o f n i c o t i n a m i d e might be antagonistic to the f o r m a t i o n of p o l y ( A D P - r i b o s e ) . (2/ Past results have shown that when nicotinic acid, nico t i n a m i d e or N A D were placed into the m e d i a at 1 x 10 4 M, large a m o u n t s of trigonelline were synthesized a n d this new synthesized trigonelline was responsible tbr the p r o m o t i o n of cell arrest in G2. '26/ It would a p p e a r that one possibility for both 7 - M X a n d 3-MB a n t a g o n i z i n g G2 arrest when present with 1 x 10 _4 M N A D was that the a d e q u a t e a m o u n t o f trigonelline to p r o m o t e G2 arrest h a d not been synthesized. C251 These experiments were performed to determine i f p o l y ( A D P - r i b o s e ) synthesis inhibitors a n d p r o m o t e r s affected G2 arrest caused by trigonelline. Some of the d a t a r e p o r t e d herein suggest that several c o m p o u n d s which are k n o w n to affect p o l y ( A D P - r i b o s y l ) a t i o n do indeed affect the ability of the root cells to respond to trigonelline. These c o m p o u n d s m a y also alter the w a y in which N A D is m e t a b o l i z e d by excised roots. W h i l e this is not conclusive d a t a , the possibility remains, a n d is even e n h a n c e d , t h a t p o l y ( A D P - r i b o s e ) might be involved in G2 arrest caused by trigonelline. In o r d e r to further investigate this possibility, e x p e r i m e n t a t i o n must be performed on trigonelline treated meristem nuclei, which directly measures the a m o u n t of p o l y ( A D P - r i b o s e ) in the nucleus and which also d e m o n s t r a t e s the b i n d i n g of poly(ADP-ribose) to a c c e p t o r proteins in these nuclei.

471

Acknowledgements This research has been supported by grant l-R15 GM 36008-01 from the National Institutes of General Medical Sciences to William A. Tramontano. The authors are grateful to Dr Lance S. Evans for his critical review of the manuscript and assistance with the data analysis. The authors also thank Mrs Marie Klasser for the excellent typing of the manuscript.

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