effects of fluorinated pyrimidines on the growth of excised pea

13
Plant & Cell Physiol., 11, 259-271 (1970) Effects of fluorinated pyrimidines on the growth of excised pea embryos l K. PARANJOTHY 2 and V. RAGHAVAN School of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia (Received October 27, 1969) The effects of some fluorinated pyrimidines on the growth of excised pea embryos {Pisum sativum var. Alaska) in sterile culture were studied. Even the lowest concentrations of the compounds tested inhibited growth in length of the embryos. In order of decreasing activity, the compounds tested were : 5-fluorodeoxyuridine, 5-fluoro- deoxycytidine, 5-fluorouridine, 5-fluoroorotic acid and 5-fluorouracil. Inhibition of growth in length of the root primordia was found to be mainly due to inhibition of cell division with no effect on cell elongation. Reversal of fluoropyrimidine induced inhibition of growth by pyrimidine bases and their related metabolites indicated that the analogues primarily inhibited DNA synthesis. Current interest in plant growth and development has led to an examination of the distribution and metabolism of DNA and RNA in plant tissues. Some of these studies have been undertaken using analogues of nucleic acid bases which interfere selectively with one or more steps in the biosynthesis of RNA and DNA. A few of the most extensively employed analogues of nucleic acid bases belong to the group of fluorinated pyrimidines in which the hydrogen atom attached to the fifth carbon of the pyrimidine ring is replaced by a fluorine atom. The mode of action and metabolism of fluorinated pyrimidines have been extensively investigated in animal tissues and bacterial cells (1, 2). They exert their various biological and biochemical effects by interfering with DNA synthesis leading to inhibition of activity of the enzyme, thymidylate synthetase (for example, FLU and FDUr), and/or by interfering with RNA synthesis by incorporation into functional RNA (for example, FLU). No comparative studies on the mode of action of the analogues have been undertaken with plant tissues. Although fluorinated pyrimidines lack the specificity of other well known inhibitors of nucleic acid synthesis like actinomycin D, puromycin, mitomycin, etc., both FLU and FDUr have been used to some extent in studying nucleic acid metabolism in plants. Recent studies on the metabolic processes underlying hormone-induced cell elongation in higher plants (3-11) and two-dimensional growth in fern gametophytes (12), for example, illustrate the use of these compounds as tools of research in plant growth and development. Among other studies in which FLU and FDUr have been successfully employed to determine the relationship Abbreviations : FLU, 5-fluorouracil; FLO, 5-fluoroorotic acid; FLUr, 5-fluorouridine; FDUr, 5-fluorodeoxyuridine; FDCy, 5-fluorodeoxycytidine; DNA, deoxyribonucleic acid; RNA, ribonucleic acid. 1 Part of a thesis submitted by the senior author for the degree of M.Sc. of the University of Malaya. 2 Present address : Department of Botany, University College of Wales, Aberysrwyth, U.K. 259 Downloaded from https://academic.oup.com/pcp/article-abstract/11/2/259/1873261 by guest on 04 February 2018

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Page 1: Effects of fluorinated pyrimidines on the growth of excised pea

Plant & Cell Physiol., 11, 259-271 (1970)

Effects of fluorinated pyrimidines on the growthof excised pea embryosl

K. PARANJOTHY2 and V. RAGHAVAN

School of Biological Sciences, University of Malaya,Kuala Lumpur, Malaysia

(Received October 27, 1969)

The effects of some fluorinated pyrimidines on the growth of excised pea embryos{Pisum sativum var. Alaska) in sterile culture were studied. Even the lowest concentrationsof the compounds tested inhibited growth in length of the embryos. In order ofdecreasing activity, the compounds tested were : 5-fluorodeoxyuridine, 5-fluoro-deoxycytidine, 5-fluorouridine, 5-fluoroorotic acid and 5-fluorouracil. Inhibition ofgrowth in length of the root primordia was found to be mainly due to inhibition of celldivision with no effect on cell elongation. Reversal of fluoropyrimidine inducedinhibition of growth by pyrimidine bases and their related metabolites indicated thatthe analogues primarily inhibited DNA synthesis.

Current interest in plant growth and development has led to an examinationof the distribution and metabolism of DNA and RNA in plant tissues. Some ofthese studies have been undertaken using analogues of nucleic acid bases whichinterfere selectively with one or more steps in the biosynthesis of RNA and DNA.A few of the most extensively employed analogues of nucleic acid bases belong tothe group of fluorinated pyrimidines in which the hydrogen atom attached to thefifth carbon of the pyrimidine ring is replaced by a fluorine atom.

The mode of action and metabolism of fluorinated pyrimidines have beenextensively investigated in animal tissues and bacterial cells (1, 2). They exerttheir various biological and biochemical effects by interfering with DNA synthesisleading to inhibition of activity of the enzyme, thymidylate synthetase (for example,FLU and FDUr), and/or by interfering with RNA synthesis by incorporation intofunctional RNA (for example, FLU). No comparative studies on the mode of actionof the analogues have been undertaken with plant tissues.

Although fluorinated pyrimidines lack the specificity of other well knowninhibitors of nucleic acid synthesis like actinomycin D, puromycin, mitomycin,etc., both FLU and FDUr have been used to some extent in studying nucleic acidmetabolism in plants. Recent studies on the metabolic processes underlyinghormone-induced cell elongation in higher plants (3-11) and two-dimensionalgrowth in fern gametophytes (12), for example, illustrate the use of these compoundsas tools of research in plant growth and development. Among other studies in whichFLU and FDUr have been successfully employed to determine the relationship

Abbreviations : FLU, 5-fluorouracil; FLO, 5-fluoroorotic acid; FLUr, 5-fluorouridine; FDUr,5-fluorodeoxyuridine; FDCy, 5-fluorodeoxycytidine; DNA, deoxyribonucleic acid; RNA, ribonucleicacid.1 Part of a thesis submitted by the senior author for the degree of M.Sc. of the University of Malaya.2 Present address : Department of Botany, University College of Wales, Aberysrwyth, U.K.

259

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260 K. PARANJOTHY and V. RAGHAVAN Vol. 11 (1970)

between nucleic acid metabolism and growth may be mentioned the requirementfor RNA and DNA synthesis during photoperiodic induction of flowering in plants(13-17), inhibition of chlorophyll synthesis (18) and morphogenesis in Acetabularia(19). The effects of other fluorinated pyrimidines like FLO, FLUr and FDCy onplant tissues are relatively unknown.

Surprisingly little work has been done on the role of nucleic acids during growthof plant embryos. Mature embryos of angiosperms are generally enclosed in theseed coat and surrounded by the nutritive tissue of the cotyledons and endosperm.Embryos can be excised from mature seeds and grown aseptically in culture withrelative ease. In many species, a few hours after soaking or after excision andculture, the embryo begins to grow by a process involving both cell division and cellelongation (20-23). Since cell division involves nuclear division it is obvious thatit is dependent upon DNA synthesis. Recent work on plant hormone action andcell elongation, on the other hand, has shown that RNA and protein synthesis arerequired for this process. Since both cell division and cell elongation occur in thesame material within a brief span of time, excised and cultured embryos appear toprovide an ideal system to study these processes and their control by appropriateinhibitors of RNA and DNA synthesis. In addition, embryos also offer theadvantage of a simultaneous analysis of the effects of the test substances on thegrowth of the root, shoot and hypocotyl.

Experiments described in this work were designed to determine the effects offluorinated pyrimidines on the growth of pea embryos in sterile culture. Fivefluorinated pyrimidines some of them well known for their specificity and selectivityhave been used. They are : FLU, FLO, FLUr, FDUr and FDCy. Attempts havebeen made to correlate the observed effects of analogues on the growth of embryoswith cell division, cell elongation and cell number of the treated tissues. Reversal ofgrowth inhibition due to analogues by pyrimidine bases or their derivatives hasgiven some indication of the metabolic blocks induced by fluorinated pyrimidines.Pea embryos were selected for experimental purposes because of the ease of excisionand culture, and the availability of information on the physiology of growth of theseedlings in relation to light and growth substances.

Material and methods

Culture methods

Seeds of Pisum sativum var. Alaska (obtained from Ferry Morse Seed Co.,Mountain View, California) were soaked in running tap water for 18 hr, surface-sterilized for 5 min in 50% 'Clorox' (a commercial hypochlorite bleach) and washedrepeatedly in sterile distilled water. Excision of the embryos from the seeds forculture was carried out aseptically in a transfer room previously illuminated byUV sterilamps. The seed was cut open with the help of a surgeon's blade to separatethe two cotyledons. Since the cotyledons are attached to the embryos the lattercan be separated at this stage by careful manipulation. The excised embryos wererinsed briefly in sterile distilled water and blotted dry before transfer to the medium.About 5 or 6 embryos were transferred to 25 ml of solidified nutrient agar mediumcontained in 9 cm petri dishes.

The basal medium used in this study consisted of macronutrient salts, trace

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Fluorinated pyrimidines and growth of embryos 261

elements, vitamins and sucrose in proportions used by RAGHAVAN and TORREY(24) for culture of Capsella embryos.

All components of the medium were mixed together and sterilized for 15 minat 15 lb/in2 (1.05 kg/cm2) in an autoclave. Analogues and reversers were coldsterilized through Millipore filters (Millipore Filter Corp., Bedford, Massachusetts,U. S. A.) and added to the autoclaved medium.

The cultures were kept in an incubator at 25±1°C which was kept completelydark except for brief periods of opening for examination. Petri dishes showingbacterial or fungal contamination were discarded.

Growth measurements

Embryos were removed from the culture medium and growth in length of theroot, hypocotyl and shoot was separately measured. Shoot was operationallydefined as the portion above the point of cotyledonary attachment, hypocotyl asthe portion between the point of cotyledonary attachment and the origin of roothairs and root as the portion below the hypocotyl. Unless otherwise specified,growth measurements were made to the nearest 0.5 mm at the end of 7 days.

Histological studies

For histological studies roots were excised and fixed by vacuum infiltration inNAVASHIN'S solution for 4 hr. They were then washed in running tap water for4—6 hr, dehydrated in a tert-h\xty\ alcohol series and infiltrated with paraffin waxand embedded. Longitudinal sections 10 y, thick were cut and stained in safraninand fast green. Cortical and epidermal cell lengths in the root sections weremeasured with an oculometer.

Mitotic and cell counts

For mitotic studies, 1.5-2.0 mm long root tips were sampled at frequent intervalsand fixed in CARNOY'S solution for 30 min. They were hydrolysed in 1 N HC1 at60°C for 10 min and stained in basic fuchsin for 2 hr in the dark. Squashes wereprepared in 45% acetic acid. Mitotic indices were computed from counts of about1000 cells per root. The maceration method of BROWN and BROADBENT (25) wasused to estimate the number of cells per root.

Results

Growth of excised pea embryos in the basal medium

Fig. 1 illustrates a time-course study of growth in length of the root, shoot andhypocotyl of excised pea embryos in the basal medium over a 7 day culture period.Measurements of the root and hypocotyl were made beginning the second day ofculture as there was no external differentiation between the two organs before thistime. It is seen from the figure that growth of the root was most rapid between thethird and sixth day after which there was a decrease in growth rate. A maximumroot length of about 35 mm was attained in 7 days. Lateral roots were not producedduring the culture period (Fig. 2). The shoot attained a length of about 15 mmover the same period. Hypocotyls attained a maximum length of about 7 mmin 3 days after which there was no further growth.

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262 K. PARANJOTHY and V. RAGHAVAN Vol. 11 (1970)

Fig. 1. Growth of excised pea embryos in the basal medium. Eachpoint represents the average of at least 30 measurements. Verticalbars=2x standard error.

Fig. 2. Excised pea embryos grown in the basal medium for 7 days.The scale is in mm and cm.

Inhibition of growth by fluorinated pyrimidines

In Fig. 3 are shown the effects of a range of concentrations FDUr, FDCy, FLUr,FLU and FLO on the growth in length of the root, hypocotyl and shoot of culturedembryos. It is seen that supplementing the basal medium with even a low con-

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Fluorinated pyrimidines and growth of embryos 263

20-

-10 -9 - 4 - 3- 7 - 6 - 5Concentration [ M x 10]

Fig. 3. Effects of fluorinated pyrimidines on the growth of the root,hypocotyl and shoot of excised pea embryos. Growth is expressed aspercentage of the control (basal medium).

centration of any of the analogues lead to a rapid decrease in the growth of embryos.Generally, with all compounds tested, root and shoot elongation was severely affectedwhile hypocotyl elongation suffered least.

Of the compounds tested, FDUr and FDCy appeared to be the most effectivecompounds in inhibiting growth of embryos. FLUr and FLO were intermediatein their growth inhibitory effects, while FLU was the least inhibitory.

A distinct morphological effect of relatively high concentrations of the analogueson the embryos was the production of lateral roots. The laterals were nearlytransparent, 1-5 mm long and about 0.5 mm in diameter (Fig. 4). Their frequencyon each root varied from 1 to 5. The appearance of lateral roots when the growthof the main root is suppressed suggests an inhibition of lateral root formation bythe growing main root tip. This is in agreement with the root decapitationexperiments of TORREY (26).

Analysis of growth inhibition

The observed inhibition of growth in length of the embryos by fluoropyrim-idines can result from an inhibition of cell division or an inhibition of cell elonga-tion or both. In further work, the effects of the test substances on cell division

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264 K. PARANJOTHY and V. RAGHAVAN Vol. U (1970)

2 0

Fig. 4. Excised pea embryos grown in 4.1 x 10~8 M FDUr for 7 days.Note formation of lateral roots. The scale is in ram and cm.

and cell elongation in the roots of cultured embryos were evaluated.Concentrations of analogues causing 80-90% inhibition of growth of the roots

were used in these studies; these are : FLU 7.7 x 10"5 M; FLO 7.7 x 10"6 M; FLUr9.5X10- 6 M, and FDCy and FDUr 1.2X10"7M. AS shown in Table 1, at theseconcentrations of analogues, growth in length of the root ceased in about 3 days.A significant difference in lengths of root growing in the basal medium and in thebasal medium supplemented with the analogue was also first observed on the thirdday of culture.

Effect of analogues on cell division

Preliminary experiments indicated that separation of cotyledons from embryosled to a rapid decrease in cell divisions in root tips that lasted approximately 12 hr.VAN'T HOF (27) reported a similar decrease in mitotic index when root tips wereexcised from pea seedlings. In view of this, excised embryos were left in the basal

Table 1Growth in length of the root of excised pea embryos in different media *

Days Basal medium FDUr FDCy FLUr FLU FLO(1 .2X10" 7 M) ( 1 . 2 X 1 0 - ' M ) (9 .5X10" 8 M) (7 .7X10" 5 M) ( 7 . 7 X 1 0 " 6 M )

12

3

4

5

a

3.6±0.37.6±1.0

15.4±1.822.8±1.0

Length mm ±

2.6±0.13.3±0.35.0+0.25.1 ±0.15.1+0.3

standard error.

2.5±0.3.3±0.5.1±0.5.1+0.5. l±0.

23

21

2

2.4+0.13.4±0.25.0+0.25.1 ±0.15.1±0.3

2.5+0.13.3±0.35.1 ±0.24.9±0.15.0±0.2

2.6±0.23.5±0.25.1 ±0.25.2+0.15.2±0.3

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Fluorinated pyrimidines and growth of embryos

4r

265

12 7224 48Hours

Fig. 5. Effect of FDUr (1.2 X 10"7 M) and FLU (7.7 x 10"5 M) on themitotic index of root primordia of excised pea embryos.

medium for 24 hr before transfer to media containing analogues or to fresh basalmedium.

The mitotic counts from root tips of embryos grown in this way in mediacontaining FDUr and FLU are presented in Fig. 5; similar curves were obtainedwith the other compounds tested. It is seen that the analogues caused a gradualarrest of mitosis in root tips. After exposure to the analogues for 48 hr there wasvirtually a complete absence of cell divisions in the root apex. Of interest is thefact that although cell division ceased by 48 hr, the root primordia grew in lengthto some extent in the media containing analogues during the first 3 days.

Cell numbers in the roots of the embryos growing in the basal medium with andwithout the analogues were also estimated. For this purpose, embryos wereincubated in basal medium for 24 hr before transfer to analogue or to fresh basalmedium and cell numbers in the root tips determined at intervals. Results (Fig. 6)

o 24 72 9648Hours

Fig. 6. Effect of FDUr (1.2 X 10"' M) and FLU (7.7 x 10"5 M) on theincrease in cell number in the root primordia of excised pea embryos.Vertical bars=2x standard error.

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Page 8: Effects of fluorinated pyrimidines on the growth of excised pea

266 K. PARANJOTHY and V. RAGHAVAN Vol. 11 (1970)

Table 2Lengths of cortical and epidermal cells in the roots of excised pea

embryos grown in different media for 3 days "

Lengthfromrootapex

0 mm

1 mm

2 mm

3 mm

CorticalEpidermal

CorticalEpidermal

CorticalEpidermal

CorticalEpidermal

m B e d ? l

15.6±2.614.7+0.3

27.4+1.631.3 + 1.7

41.3 + 2.238.8±1.5

54.0 + 1.655.6±0.8

FDUr

10"'M)

14.2±0.614.7±0.4

27.7 + 1.928.0 + 2.1

40.1+2.039.0±2.1

58.8±3.651. 1±4.3

FDCy

10-'M)

14.9+0.516.5±0.4

26.9 + 2.226.0+2.2

41.1+2.640.5 + 3.0

52.6+3.354.3±1.7

FLUr

10-«M)

15.5±0.617.0±0.7

30.4±5.631.3 + 2.6

42.1+3.437.3±3.8

58.7±4.157.1 ±5.7

FLU(7.7x

1 0 " 5 M )

13.5±0.615.0±0.9

34.8±4.132.2±3.0

39.9±3.240.5±3.5

59.0±5.052.4±4.6

FLO(7.7x

lO-o M)

14.8±0.716.2±0.8

28.9±2.229.4±2.8

41.5±2.442.7±2.5

52.2±3.057.8±1.3

" Length fx ± standard error.

showed increase in cell numbers ceased by about the second day in the treatedembryos. These data confirm the results obtained from mitotic counts and excludepossible periodic division cycles that might have been missed between the samplingtimes for mitotic counts.

Effect of analogues on cell length

In this experiment excised embryos were grown in different media for 3 days.Cell lengths at various regions of the root were measured beginning from the roottip (designated as 0 mm in Table 2). As seen from Table 2, lengths of epidermaland cortical cells were not significantly different in roots excised from control andtreated embryos. It thus appears that inhibition of cell elongation is not a majorfactor in inhibition of growth of the embryos induced by fluoropyrimidines.

Reversal of analogue induced inhibition of growth with pyrimidine compounds

The studies described above indicate that fluoropyrimidines at the concentra-tions used inhibit growth of excised pea embryos primarily by inhibiting celldivision, with no appreciable effect on cell elongation. These results appear to beconsistent with their inhibitory effect on DNA synthesis (2, 28). Further informa-tion on the mode of action of these compounds was sought from an analysis of theeffects of pyrimidine bases and related compounds in reversing the growth inhibitioninduced by the analogues.

Results are presented in Table 3. It is seen that inhibition of growth of theroot and shoot systems of cultured embryos induced by fluorinated pyrimidines waseffectively reversed by thymidine or thymidylic acid. Generally, on a molar basis,thymidine was more effective than thymidylic acid. When FLUr (9.5 X 10"8 M)was the inhibitor used, relatively high concentrations of uridine (2x 10~3 M), cytidine( 2 X 1 0 " 3 M ) , uridylic acid (1 .5X10- 3 M) , cytidylic acid (1 .5X10" 3 M) and oroticacid (3.2 X 10~3 M) completely reversed the inhibition. FLU (7.7 x 10"5 M) inducedgrowth inhibition was completely reversed by high concentrations of thymine

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Page 9: Effects of fluorinated pyrimidines on the growth of excised pea

NoneThymineThymidineThymidylicacidUracilUridine

1.9xlO'3

6.2xlO"5

1.6x10"'

4.5xlO"3

2.0X10"3

—-

tttt

tttt

_

4+

— (+r)

- (++)tttt

tttt

/ 1 1 \

— \T~r/tttt

tttt

- (+0W(tttt)

-H-(tttt)

ttlt

-wm-Ht(tttt)

•H-(tW)

+(ttt)- ( -H- )

-(++)+ (»)-W

-M

+(»)

- ( • H - )

- ( • H - )

tttt

tttt

- (w)-(++)- (++)-(+tt)

- ( -H)

Fluorinated pyrimidines and growth of embryos 267

Table 3Reversal of fluoropyrimidine-induced inhibition of root and shoot growth in

cultured embryos by pyrimidim bases and related compounds

~ Concentration FDUr FDCy FLUr FLU FLOKeversers ( M ) ( 1 . 2 X 1 0 " ' M ) ( 1 . 2 X 1 0 " ' M ) ( 9 . 5 X 1 0 " 6 M ) ( 7 . 7 X 1 0 " 5 M ) (7.7X10-<>M)

titt

Uridylic acid 1.5X10"3

Cytosine 4.5xlO"3 - iCytidine 2.0xl0"3 ++

Cytidylic !. 5x io-3acidOrotic acid 3.2xlO"3

The concentrations of reversers given above were found to produce optimal reversal of growthinhibition. Reversal is indicated in relative units as percentage of growth in the basal medium,as follows: - 0 - 2 0 % ; +20-40%; +h 40-60%; m 60-80%; tttt 80-100%. Values for reversalof inhibition of shoot growth are indicated in parenthesis, if different from that of root.

(1.9X10~3M). Partial, nonspecific reversal of root and shoot growth inhibitionwas also observed by high concentrations of uracil, cytosine, uridine, cytidine,uridylic acid, cytidylic acid and orotic acid.

Complete reversal of fluoropyrimidine-induced growth inhibition by lowconcentrations of thymidine or thymidylic acid suggested that the analoguesprimarily inhibited DNA synthesis. Although precursors of RNA like uridine,uridylic acid and orotic acid also reversed some of the fluoropyrimidine-inducedgrowth inhibitions, these compounds were active at relatively high concentrations.The structural similarities between analogues and the reversers and the relativelyhigh concentrations of the latter, required for complete reversal suggested acompetitive type of growth reversal.

The competitive nature of growth reversal was tested by determining theefficacy of optimal concentrations of pyrimidine compounds (already determinedin the reversal studies described above) in reversing growth inhibition produced byincreasing concentrations of analogues. The results for reversal of root and shootgrowth inhibitions produced by increasing concentrations of FLUr with thymidine,uridine and orotic acid and FLU with thymine and thymidine are summarised inTable 4.

Thymidine, uridine and orotic acid completely reversed root growth inhibitionsproduced by 9.5X10~6M of FLUr. Inhibition of root growth produced by1.9 X 10"s M of FLUr was completely reversed by nearly the same concentration ofthymidine but only partially by uridine and orotic acid. Root growth inhibitionsproduced by still higher concentrations of FLUr (3.8X10~5M) were partiallyreversed by thymidine while uridine and orotic acid were ineffective. It appears

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268 . K. PARANJOTHY and V.. RAGHAVAN Vol. 11 (1970)

Table 4Reversal of growth inhibitions due to increasing concentrations of FLUr

and FLU by thymidine, uridine, orotic acid and thymine

Root

FLUr

FLU

Shoot

FLUr

FLU

Concentrationof analogues

(M)

0 (Basal medium)9.5x10-"1.9X10"5

3.8xlO"6

7.6xI0"5

0 (Basal medium)7.7xl0"5

1.5xlO"s

3.9x10-'

0 (Basal medium)9.5X10"6

1.9X10"5

3.8xlO"6

7.6xlO-s

0 (Basal medium)7.7xlO"5

1.5x10-*3.9x10-"

Thymidine(6.2x

10"5M)

36.1 + 1.35.1±1.

36.0±l.9.3+0.5.8±0.

35.8 + 1.15.8±0.

2

,2

,3

5

3

0

8

8.9±0.85.8+0.

15.3±1.15.2±0.9.8+0.9.3±0.3.3±0.

15.6±1.14.8±0.8.0±0.2.5+0.

2

1

8

5

5

1

2

6

6

1

Uridine(2. Ox

10-31

35.8±1.34.3±1.9.3±0.3.5±0.3.5±0.

———

14.9 + 1.14.8±0.4.5±0.3.0+0.2.5+0.

a)

1

1

5

1

1

0

5

2

1

1

Orotic acid(3.2x

10"3M)

36.0±1.536.1 + 1.510.8±1.03.8+0.23.6±0.2

_——

15.0 + 1.114.3+0.65.6±0.34.1 ±0.12.8±0.1

Thymine(1.9X

10"3M)

34.9±1.33.8+1.5.0+0.4.5±0.

————

15.6±1.15.1+0.3.1+0.2.4±0.

2

3

3

2

1

8

1

1

None(analogues

only)

4.5±0.24.1 ±0.24.1+0.34.2±0.1

5.0±0.25.1 ±0.24.2+0.1

7.6±0.43.5±0.33.1±0.23.0±0.2

10.1 ±0.44.3±0.32.5±0.1

The last column refers to growth of root and shoot in media containing analogues only.Length mm ^ standard error.

Table 5Effects of pyrimidine bases and related compounds on the growth in length

(mm±standard error) of root and shoot regionsof excised pea embryos

Basal mediumCytosineCytidineCytidylic acidUracilUridineUridylic acidThymineThymidineThymidylic acidOrotic acid

Concentration(M)

2.3x10-'l.OxlO"5

7.7x10-*2.2xlO-3

1.0x10-37.7xl0-4

1.9x10-38.3x10-31.6x10-'3.2x10-3

Root

33.2±1.817.3 + 1.3

29.1 ± 1.430.5 + 1.223.9±1.429.4±1.833.5±1.030.1 ± 1.532.8+1.231.3±0.930.8+1.1

Shoot

15.3±1.115.4±0.915.0±0.614.9±0.713.9±0.914.7±0.715.1 ±0.613.2 + 1.015.1+0.815.2+0.713.9+0.8

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Fluorinated pyrimidines and growth of embryos 269

from these results that thymidine is more efficient than either uridine or orotic acidin reversing growth inhibitions produced by increasing concentrations of FLUr.A similar trend was observed for reversal of shoot growth inhibitions produced byincreasing concentrations of FLUr with thymidine, uridine and orotic acid. Rootand shoot growth inhibitions produced by increasing concentrations of FLUsimilarly were more easily reversed by thymidine than by thymine. These resultsindicate that as compared to reversal of FLUr-induced growth inhibition byuridine and orotic acid, and FLU-induced growth inhibition by thymine, reversalby thymidine of growth inhibitions induced by FLUr and FLU may be of a non-competitive nature.

Effects of pyrimidine compounds on root and shoot growth

The effects of the reversers themselves in the absence of analogues on thegrowth of root and shoot are summarised in Table 5. In this experiment con-centrations of the reversers causing optimal reversal of growth inhibitions inducedby FLU (7.7X10"BM) and FDUr (1.2X10"7

M) were used. Only cytosine anduracil produced a significant inhibition of root growth at concentrations of 2.3 X 10"3

M and 2.2X10" 3 M respectively. Shoot growth was not affected by any of thecompounds tested. There was no significant promotion of growth of the root orshoot by the reversers at the concentrations used, thus indicating that reversal offluoropyrimidine-induced growth inhibitions by the pyrimidines was not due totheir growth promoting effects per se. The results do not however rule out thepossibility of reversal resulting from the prevention of entry of analogues into thecells by the pyrimidine compounds.

Discussion

The main conclusion from this study is that none of the fluorinated pyrimidinestested promote growth of cultured pea embryos, while even relatively low concentra-tions of the compounds inhibit their growth. Shoots generally required a higherconcentration of the analogues than roots for the same degree of inhibition. Thisdifference in the sensitivity of the two organs may be due to the fact that roots growin direct contact with the medium while shoots grow upright away from the medium.NEUMANN (7) showed that the decreased inhibition of nucleic acid synthesis byactinomycin D in shoots of germinating lettuce seedlings which were in contact withthe drug through the roots-was due to its decreased uptake into the shoot. It is alsopossible that by the time the analogues exert their inhibitory effects on the root,the functions of the latter are impaired and movement of the analogues into theshoots prevented.

It is observed in this study that root primordia of excised embryos transferreddirectly to media containing analogues normally increased their length up to 3 days(Table 1). During this period, only few mitotic divisions were observed (Fig. 5)and extension growth appeared to be mainly due to cell elongation. Our unpublish-ed results have shown that protein synthesis in the treated roots declined considerablyduring this period. Since protein synthesis is essential for cell elongation (3, 29)and the bulk of RNA in expanding cells is formed prior to the main period of cellelongation (6), it is possible that proteins required for cell elongation were already

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270 K. PARANJOTHY and V. RAGHAVAN Vol. 11 (1970)

synthesised before the analogues exerted their inhibitory effects in the embryos.WALTON (22) has reported that at 26°C excised embryonic axes of Phaseolus vulgarisbegan elongation after a 7 hr incubation in buffer or water. Both RNA and proteinsynthesis began prior to the initiation of axis elongation and cell elongation wasfound to precede cell division by 4 or 5 hr. Cell expansion prior to cell divisionduring germination has also been reported for barley (20), Viciafaba (23) and Vignasesquipedalis (21). It is also possible that the analogues did not inhibit proteinsynthesis required for cell elongation or that proteins required for cell elongationwere available from the reserves already present in the cells at the time of germina-tion.

Reversal of fluoropyrimidine induced inhibition of growth of pea embryos byrelatively low concentrations of thymidine and thymidylic acid indicates that theanalogues primarily inhibit DNA synthesis. However, the partial reversal of FLUinduced root growth inhibition by thymidine and its complete reversal by thyminesuggest that the analogue may inhibit metabolic processes besides DNA synthesis.As mentioned earlier in this paper, FLU is known to inhibit RNA synthesis as well.According to KEY (4) the inhibitory action of this analogue is confined to thesynthesis of soluble and ribosomal RNA without affecting D-RNA synthesis. Itis possible that FLU inhibited RNA synthesis in pea embryos to some extent,although RNA precursors such as uracil, cytosine and orotic acid were found to beonly very slightly effective in reversing FLU action. The present experimentsfurther indicate that reversal of FLU-induced growth inhibition by thymine is of acompetitive nature in contrast to the non-competitive mode of reversal by thymidine.The close structural resemblance between FLU and thymine (5-methyluracil)would allow the mutual competition. The possibility that FLU inhibits growthof shoot and root by interfering with different facets of macromolecular synthesisin the embryos deserves to be investigated.

It is interesting to note that the analogues failed to inhibit cell elongation whileinhibiting DNA synthesis. NOODEN (10) found that FDUr failed to inhibit thegrowth of corn coleoptile sections or artichoke tuber discs over a 20 hr period withor without auxin. These observations do not support the view of NITSAN and LANG(8, 9) who found a requirement for DNA synthesis in cell elongation.

References

(7) BROCKMAN, R. W. and E. P. ANDERSON: Pyrimidine analogues. In Metabolic Inhibitors. I.p. 239-285. Edited by R. M. HOCHSTER and J. H. QUASTEL. Academic Press, N. Y., 1963.

( 2 ) HEIDELBERCER, C.: Fluorinated pyrimidines. In Progress in Nucleic Acid Research and Molecular

Biology, p. 1-^9. Edited by J. N. DAVIDSON and W. E. COHN. Academic Press, N. Y., 1965.(3) KEY, J. L.: Ribonucleic acid and protein synthesis as essential processes for cell elongation.

Plant Physiol., 39, 365-370 (1964).( 4) KEY, J. L.: Effect of purine and pyrimidine analogues on growth and RNA metabolism in

the soybean hypocotyl - the selective action of 5-fluorouracil. ibid., 41, 1257-1264 (1966).( 5) KEY, J. L. and J. INGLE : Requirement for the synthesis of DNA-Iike RNA for growth of excised

plant tissue. Proc. Nail. Acad. Sri. U. S., 52, 1382-1388 (1964).( 6) MASUDA, Y., G. SETTERFIELD and S. T. BAYLEY: Ribonucleic acid metabolism and cell expansion

in oat coleoptile. Plant & Cell Physiol., 7, 243-262 (1966).

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Fluorinated pyrimidines and growth of embryos 271

(7) NEUMANN, J.: The effect of actinomycin D on lettuce seedlings and its differential uptake byroots and shoots. Physiol. Plant., 17, 363-370 (1964).

( 8) NITSAN, J. and A. LANG: Inhibition of cell division and cell elongation in higher plants byinhititors of DNA synthesis. Develop. Biol, 12, 358-376 (1965).

( 9 ) NITSAN, J. and A. LANG : DNA synthesis in the elongating non-dividing cells of the lentil epicotyland its promotion by gibberellin. Plant Physiol., 41, 965-970 (1966).

(10) NOODEN, L. D.: Studies on the role of RNA synthesis in auxin induction of cell enlargement.ibid., 43, 140-150 (1968).

(11) SETTERFIELD, G.: Growth regulation in excised slices of Jerusalem artichoke tuber tissue.Symp. Soc. Exptl. Biol., 17, 98-126 (1963).

(12) BOPP, M.: Control of differentiation of fern-allies and bryophytes. Ann. Rev. Plant Physiol.,19, 361-380 (1968).

(13) BONNER, J. and J. A. D. ZEEVAART: Ribonucleic acid synthesis in the bud an essential componentof floral induction in Xanthium. Plant Physiol., 37, 43-49 (1962).

(14) CHERRY, J. H. and R. VAN HUYSTEE: Effects of 5-fluorouracil on photoperiodic induction andnucleic acid metabolism of Xanthium. ibid., 40, 987-993 (1965).

(15) SALISBURY, F. B. and J. BONNER: Inhibition of photoperiodic induction by 5-fluorouracil.ibid., 35, 173-177 (1960).

(16) UMEMURA, K. and Y. OOTA : Effects of nucleic acid- and protein-antimetabolites on frond andflower production in Lemna gibba G3. Plant & Cell Physiol., 6, 73-85 (1965).

(17) ZEEVAART, J. A. D.: DNA multiplication as a requirement for expression of floral stimulusin Pharbitis nil. Plant Physiol., 37, 296-304 (1962).

(18) KIRK.J. T. O. andR. A. E. TILNEY-BESSETT: Theplastids: Their Chemistry, Structure, Growthand Inheritance. W. H. Freeman & Co., London, 1967.

(19) DE VITRY, F.: Etude de l'action de la 5-fluorodeoxyuridine sur la croissance et la morphog6nesed'Acetabularia mediterranea. Exptl. Cell Res., 25, 697-699 (1961).

(20) CALDECOTT, R. S. and L. SMITH: A study of X-ray-induced chromosomal aberrations inbarley. Cytologia, 17, 224-242 (1952).

(21) OOTA, Y.: A study on the relationship between water uptake and respiration of isolated beangerm-axes. Physiol. Plant., 11, 710-721 (1958).

(22) WALTON, D. C : Germination of Phaseolus vulgaris I. Resumption of axis growth. PlantPhysiol., 41, 298-302 (1966).

(23) WOLFF, S.: Some aspects of the chemical protection against radiation damage to Vina fabachromosomes. Genetics, 39, 356-364 (1954).

(24) RAGHAVAN, V. and J. G. TORREY: Growth and morphogenesis of globular and older embryosof Capsella in culture. Amer. J. Bot., 50, 540-551 (1963).

(25) BROWN, R. and D. BROADBENT: The development of cells in the growing zones of the root.J. Exptl. Bot., 1, 249-263 (1950).

(26) TORREY, J. G.: The induction of lateral roots by indoleacetic acid and root decapitation.Amer. J. Bot., 37, 257-264 (1950).

(27) VAN'T HOP, J.: Cell population kinetics of excised roots of Pisum sativum. J. Cell Biol., 27,179-189 (1965).

(28) KIHLMAN, B. A.: Actions of chemicals on dividing cells. Prentice-Hall, Inc., EnglewoodCliffs, New Jersey, 1966.

(29) NOODEN, L. D. and K. V. THIMANN: Evidence for a requirement for protein synthesis forauxin-induced cell enlargement. Proc. Natl. Acad. Sci. U. S., 50, 194-200 (1963).

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