The Effect of Adenine on Growth, Starch and ADPG Content and ADPG Pyrophosphorylase Activity in Suspension-cultured Tobacco Cells

LUDMILLA v. GAMANETZ and K. Z. GAMBURG

Institute of Plant Physiology and Biochemistry, Siberian Branch, USSR Academy of Sciences, 664033 Irkutsk, USSR

Received March 23, 1981 . Accepted May 21, 1981

Summary

The starch content in tobacco cells cultured for 3 days in fresh medium with 0.1 mM adenine increased several times and growth of the culture was delayed as compared to cells cultured without adenine. Sucrose and auxin promoted the adenine effect on starch accumulation. An increase in starch content and the inhibition of culture growth were more obvious in suspen­sions with greater initial dilution of the inoculum. Under adenine influence ADPG content in the cells increased 100-fold and UDPG content fell to undetectable levels. Cells treated with adenine showed greater ADPG pyrophosphorylase activity than the controls. An addition of adenine to the reaction mixture also caused stimulation of the enzyme activity. Adenine is pre­sumed to raise the starch content in the cells through the stimulation of ADPG biosynthesis. Growth inhibition caused by adenine may be related to the reduction of UDPG pool.

Key words: Nicotiana tabacum, adenine, ADPG, ADPG pyrophosphorylase, batch suspension culture, starch.

Introduction

Adenine is sometimes incorporated in nutrient media for plant tissue and cell cul­tures, however, its effect on growth and metabolism of these cultures remains obscure. The promotion of organogenesis was reported in some callus cultures (Men­henett, 1970; Nitsch et al., 1967; Rao et aI., 1976; Thorpe and Murashige, 1970). The latter authors observed a close correlation between the induction of shoot formation and starch accumulation in the tobacco callus tissue. It is known that the starch bio­synthesis proceeds with ADPG as a preferential glucosyl donor (Turner and Turner, 1975), and it seems very likely that exogenous adenine can affect starch biosynthesis in cultured plant cells.

We have previously reported (Gamburg and Gamanetz, 1977) that adenine caused a significant increase in starch content in the suspension-cultured tobacco cells. This communication presents a more detailed study of the observed phenomenon.

Abbreviations: ADPG, adenosine-5'-(a-D-glucopyranosyl)-pyrophosphate; NAA, l-naphtha­leneacetic acid; UDPG, uridine-5'-(a-D-glucopyranosyl)-pyrophosphate.

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62 LUDMILLA V. GAMANETZ and K. Z. GAMBURG

Materials and Methods Cell culture

The callus culture was obtained from stem pith parenchyma of tobacco (Nicotiana tabacum L., cv. Trapesond) by R. G. Butenko (Institute of Plant Physiology, USSR Academy of Scien­ces, Moscow) in 1961 and kindly presented to us for experiments in 1965. The cell culture has been maintained from 1966 to the present day by weekly transfers of 3 - 4 ml grown suspension to 60 ml fresh medium. For the experiments, a stationary phase suspension (7th day of subcul­ture) was properly diluted by a fresh medium containing salts of Murashige and Skoog (1962) and other constituents (mg/I): sucrose 20,000; thiamine-HCI 0.4; pyridoxine-HCI 0.1; myo-ino­sitol80; NAA 2. Five-ml portions of suspension thus obtained were transferred to 16x 150 mm test tubes with or without adenine and cultured in a roller at 60 rpm and 26° in the dark. For the determination of the nucleoside diphosphoglucose content and ADPG pyrophosphorylase activity, the suspension was grown in 250 ml Erlenmeyer flasks (75 ml suspension in each flask) on a reciprocal shaker with 80 strokes per minute. Cells were separated from the medium by suction on a glass filter, weighed and used for analyses.

Determination 0/ starch

Soluble sugars were washed out with 80 per cent ethanol. Insoluble residue was digested with 0.2 mg/ml diastase (Merk) for 24 h at 38°C. Maltose released was determined by a ferricyanide method (Ermakov, 1972).

Determination 0/ ADPG and UDPG

Cells were homogenized in a chilled mortar with 10% HCI04 (2: 1, v/w). The brei was fil­tered through a glass filter and the residue was twice extracted with 5% HCI04• The combined extract was centrifuged 15 min at 10,000 xg and the supernatant was adjusted to pH 7 with 4 M KOH. The precipitate of KCI04 was removed by centrifugation and the solution volume was reduced to 5-10 ml in a rotor evaporator at 30°C. Then ethanol was added to get 70% ethanol in the final volume and after cooling for 12 h, the precipitate of polymers was removed by cen­trifugation. The solution was reduced in volume, passed through a column (0.9x 16 em) of Dowex lx4 200-400 mesh in formiate form and eluted with 4M HCOOH+0.8M HCOONH4• Fractions with maximal absorbance at 260 nm were combined and incubated with activated charcoal. The charcoal was eluted with the mixture of ethanol, 25 per cent NH40H and water (50 : 10 : 36). Eluate was reduced to a small volume and used for a paper chromatography with 1 M acetate buffer pH 7.5 and ethanol (30 : 75) as a solvent. UV-absorb­ing zones corresponding to ADPG and UDPG were eluted with 0.1 M HCI and measured spec­trophotometrically. No corrections for the losses during extraction and purification procedures were made.

Determination 0/ ADPG pyrophosphorylase activity

It was determined as described by Sakalo and Lobov (1978) with some modifications. Cells were homogenized in a chilled mortar with 0.05 M tris-HCI buffer pH 7.3 containing 1 mM EDT A and 10 mM reduced glutathione. Homogenate was filtered through a glass filter and cen­trifuged at 22,000 x g for 20 min. The protein fraction precipitated between 30% and 80% of saturation with ammonium sulnphate, was dissolved in 0.05 M tris-HCI buffer pH 7.3, and dia­lysed overnight against 11 of the same buffer. The dialysed preparation was assayed for ADPG pyrophosphorylase activity. The protein content of the preparations was measured by the Lowry et al. (1951) method. The reaction mixture consisted of tris-HCI buffer (pH 7.3), 10 Jlmoles; glucoso-l-phosphate, 5 Jlmoles; ATP, 2.5 Jlmoles; NaF, 10 Jlmoles; MgClz,

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Adenine effect on growth and starch content 63

10 !1moles; total volume 0.3 m!. The reaction was stopped by 30 sec boiling after 1 h incubation at 37°C. The reaction mixture was spotted on a paper and chromatographed as described above. It was shown in preliminary experiments that ADPG did not appear when the enzyme, glucoso-1-phosphate or ATP was absent in the reaction mixture.

Identification 0/ ADPG and UDPG

UDPG isolated from the tobacco cells had chromatographic properties similar to the UDPG preparation (<<Reanal», Hungary). For the identification of ADPG extracted from the tobacco cells, ADPG synthesized in ADPG pyrophosphorylase reaction was used as a chromatographic tracer. The eluate corresponding to ADPG was additionally checked for the presence of the glu­cose moiety by hydrolysis with 0.1 M HCl at 100°C and paper chromatography with n-buta­nol-acetic acid-water (4: 1 : 5, upper phase) as a solvent. Glucose was found after spraying with aniline-phthalate. The presence of adenine moiety in the eluate of ADPG spot was shown by the absence of a change in UV-absorption spectrum after the treatment with bromine (Venk­stern and Baev, 1965).

All experiments were repeated 3-4 times, the results were similar. Each variant of an experi­ment has performed with 2 - 5 replicates.

Results

The starch content in tobacco cells cultured for 3 days in fresh medium with ade­nine increased several times as compared to cells grown without adenine (Table 1).

Table I: The effect of different adenine concentrations on the fresh weight and starch content in tobacco cells cultured in suspension for 3 days (initial dilution of inoculum I : 100).

Adenine Fresh weight, Starch content mM mgper mgper Ig [!g per

I tube fresh weight I tube

0 36 1.8 61 0.01 29 4.5a 129a 0.1 16a 7.la 144a I Ila Il.Oa 120a

a - Significant at 0.05 level.

This effect was accompanied by severe inhibition of growth of the culture. At a con­centration of 1 mM adenine, the total amount of starch in the culture dropped as compared to 0.1 mM in spite of the highest content of starch calculated on the fresh weight basis. Consequently, 0.1 mM adenine was used in all subsequent experiments.

Microscopic observation of the cells stained with iodine confirmed the striking effect of adenine on the starch content: in adenine-treated cells the starch grains were more abundant, more densely stained and had a diameter 2 - 3 times greater. Similar but weaker effects were induced by adenosine and its phosphorylated derivatives. As shown in Table 2, an increase in starch content was specific for adenine because other nucleic acid bases did not cause any effects.

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64 LUDMILLA V. GAMANETZ and K. Z. GAMBURG

Table 2: The effects of some purines and pyrimidines (0.1 mM) on the fresh weight and starch con­tent in tobacco cells cultured in suspension for 3 days (initial dilution of inoculum 1 : 100).

Substances Fresh weight, Starch content, mg per 1 tube mg per Ig

fresh weight

41 1.8 Adenine 16a 5.9a Uracil 44 2.0 Guanine 24a 1.7 Cytosine 43 1.8 Thymine 42 1.6

a - Significant at 0.05 level.

The maximal stimulation of starch accumulation was observed on the third day of subculture {Table 3}. The starch content in the cells as affected by adenine diminished during the following days of cultivation. The growth of the cell culture was retarded by adenine during the whole period of subculture. It may be supposed that it takes a certain time for adenine to affect the accumulation of starch, and after 3 days, the deg­radation of starch prevails over its synthesis due to limitation of sugar supply.

Table 3: The effect of adenine (O.1mM) on growth and starch accumulation in suspension-cultured tobacco cells (initial dilution of inoculum 1 : 200).

Parameters Adenine Duration of cultivation, hours 12 24 48 72 96 168

Fresh weight, 6.3 7.9 16.1 19.7 36.9 150.2 mg per 1 tube + 4.7 6.6 6.8a 12.1a 17.1a 37.7a

Starch content, 4.6 5.1 5.5 6.8 4.6 0.8 mg per 1 g fresh weight + 4.7 6.6 18.4a 37.3a 21.6a 3.3a

Starch content, 29 40 89 134 170 120

f.lg per 1 tube + 22 44 125a 451a 369a 124

a - Significant at 0.05 level.

The starch content in the cells and adenine effect on it greatly depended on the extent of dilution of the inoculum at the beginning of the subculture. The greatest dilution possible for the tobacco cell culture without causing a measurable lagperiod was 1 : 200 {volume of grown suspension: volume of fresh medium, Vysotskaya and Gamburg, 1975}. The dilution commonly used for the maintenance of the culture was 1 : 20. As shown in Table 4, the highest starch content was observed at a 1 : 200 dilu­tion. The enhancement of starch accumulation and growth inhibition caused by ade­nine was also maximal at this dilution. No adenine effects were observed with the dilution of 1 : 20. In a dense suspension limitation of the sugar supply is supposed to

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Adenine effect on growth and starch content 65

Table 4: The dependence of the adenine effect on fresh weight and starch content in suspension­cultured tobacco cells on the initial dilution of inoculum with fresh medium.

Initial Fresh weight, mg per I tube Starch content, mg per dilution I g fresh weight

Initial Control Adenine Control Adenine After 3 days O.lmM O.lmM

1:200 2.0 12.1 3.la 8.3 74.3a I: 100 4.3 25.3 12.la 1.9 11.6a I: 50 9.4 64.6 48.0a 1.2 3.7a I: 20 19.8 160.2 160.7 0.8 0.8

a - Significant at 0.05 level.

begin early, i.e. before adenine could exert any influence. It is also possible that each cell takes up a greater amount of adenine in more diluted suspension.

In some experiments, adenine was added after a 3-day incubation of the cells with fresh medium which lacks NAA. In such cases the adenine effect on starch accumula­tion was small, but was increased by simultaneous addition of sucrose or NAA (Table 5). The greatest increase in starch content was observed with adenine, sucrose

Table 5: The effects of adenine, sucrose and NAA on starch content of suspension-cultured tobacco cells':').

Adenine, Sucrose, NAA Starch content, O.lmM 3% 2 mg/l mg per I g fresh weight

1 :200 1:100':":')

17.6 3.5 + 18.5 4.4

+ 24.5a 6.6a + 19.5 7.4a

+ + + 35.la 17.0a + + 17.9 7.la

+ + 26.9a 14.3a + + 26.6a 7.8a

,:.) The cells were incubated for 3 days with fresh medium without NAA, then, substances indi­cated were added and the starch content was determined 2 days after their addition.

':~':~) initial dilution of inoculum. a - Significant at 0.05 level.

and NAA added simultaneously. Thus, it may be concluded that the effect of adenine on the starch content of tobacco cells depends on the supply of sugar and auxin.

The UDPG content in tobacco cells grown without adenine significantly exceeded that of ADPG (Table 6). In the presence of adenine the ADPG content increased approximately IOO-fold and UDPG disappeared almost completely. An increase in ADPG content caused by adenine was more pronounced in the presence of NAA. It

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66 LUDMILLA V. GAMANETZ and K. Z. GAMBURG

corresponds to the promotion of the adenine effect on starch content by auxin (Table 5).

Table 6: The effects of adenine and NAA on the content of ADPG and UDPG in tobacco cells cul-tured in suspension for 3 days.

Experiment NAA, Adenine, ADPG, nmoles UDPG, nmoles 2 mg/l 0.1 mM perl g per 1 per 1 g per 1

fro W. flask fro w. flask

+ 0.75 0.37 11.5 5.7 2 0.45 0.21 8.5 3.9

+ 90 90 :;- ~:- :;.:;.

2 56 14 :;-:;- :;-:;-

+ + 149 57 :;.:;- :;-:;-

2 101 30 :;.::. :;.:;-

':-':- traces only.

The ADPG pyrophosphorylase activity of adenine-treated cells was nearly 2.5 tim­es higher that of the cells grown without adenine (Table 7). The activity of enzyme

Table 7: The effects of adenine and NAA on the activity of ADPG pyrophosphorylase in tobacco cells cultured in suspension for 3 days.

NAA, Ade- ADPG, ADPG, Protein of preparation, 2 mg/l nIne, nmoles per 1 g fAmoles per 1 mg fAg per 1 g

0.1 fresh weight protein fresh weigh t mM

51 85 64':-) 2.1 4.7 1.9 2.4 1.8 3.3 + 30 58 44 0.6 0.7 0.5 4.8 8.1 8.5 + 96 26 1.0 0.6 9.5 6.8 + + 260 62 3.8 1.3 10.2 6.9

,:-) Data of separate experiments.

preparations of cells grown in the presence of NAA was significantly lower as com­pared to cells grown without NAA. This difference was more contrasting when the activity was estimated on the basis of protein content because NAA increased protein content of preparations 2 - 3 times. It should be mentioned that the activity varied from one experiment to another, but each experiment revealed the influence of ade­nine and NAA.

As shown in Table 8, adenine added to the reaction mixture also increased the ADPG pyrophosphorylase activity. Thus, it may be supposed that the enhancement of ADPG pyrophosphorylase activity of adenine-treated cells is partly due to the direct effect of adenine on the enzyme.

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Adenine effect on growth and starch content 67

Table 8: The influence of adenine added to the reaction mixture on the activity of ADPG pyro­phosphorylase preparations extracted from tobacc'o cells cultured in medium with 2 mg!l NAA for 3 days.

Adenine, ADPGformed, mM nmoles per 1 mg

protein of preparation

0 420 0.1 660 0.5 730 1.0 990

The question is now whether the adenine effect on starch accumulation is specific for the tobacco cell strain used in the experiments. The examination of other cell cul­tures grown in the laboratory showed that adenine caused an increase of starch con­tent in the cultured cells of other origin too (Table 9). This may be assumed to be the general mode of adenine effect on the cultured plant cells.

Table 9: The effect of adenine 0.1 mM on the starch content in various plant cells cultured in suspension for 3 days.

Discussion

Cultures

Soybean Potato Orache (Atriplex sp.) Dewberry (Rubus idaeus)

Starch content, mg per 1 g fresh weight

Control Adenine

2.6 0.4 4.2 2.2

7.9 1.7

12.3 4.7

The data obtained show that adenine may cause an increase in starch content in cultured plant cells when incorporated into the nutrient media. Some authors observ­ed an increase of adenosine nucleotide content after the treatment of the plant cells with adenine (Anderson, 1977; Doree et aI., 1971; Merrett and Handoll, 1967). How­ever up to the present time we have not found any publication on the effect of ade­nine on the level of ADPG. The striking increase of the ADPG content in adenine­treated cells led us to the conclusion that ADPG biosynthesis in tobacco cells is limit­ed by an unsufficient size of the adenine pool and that exogenous adenine appeared to

remove this limitation. To the contrary, exogenous uracil did not cause any change in the content of UDPG in cultured tobacco cells (11.6±1.0nmoles and 12.4±O.8 nmoles per 1 g fresh weight in the control and uracil variant corresponding­ly). An increase in ADPG pyrophosphorylase activity may be an additional way of

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68 LUDMILLA V. GAMANETZ and K. Z. GAMBURG

adenine influence on the level of ADPG which is consistent with the observation of Levy and Preiss (1976) on the stimulatory action of adenosine and AMP on the enzy­me activity.

According to contemporary views ADPG is preferentially used by plant cells for starch biosynthesis (Preiss and Levy, 1979; Turner and Turner, 1975). It may there­fore be assumed that stimulation of ADPG biosynthesis is the reason of adenine­induced enhancement of starch accumulation. Adenine effect on starch accumulation may be observed only when there is no limitation of starch biosynthesis by the supp­ly of sugar. This results from the data presented in Tables 2, 3 and 4 and the data of several authors on an increase of starch content in cultured plant cells by additional sucrose in the medium (Obata-Sasamoto and Suzuki, 1978; Chu Lie-Jen and Schan­non, 1975).

Our results clearly demonstrate that auxin promoted adenine-induced increase in the content of starch and ADPG. But this action of auxin cannot be related to the influence on ADPG pyrophosphorylase activity because inhibition was observed. It may be assumed that auxin exerts its influence through the promotion of adenine uptake or transformation of adenine and sugars to the immidiate precursors of ADPG. This explanation is supported by our previous observation of the stimulation of respiration of tobacco cells by auxin (Kondrashova et aI., 1974).

An increase in ADPG and starch content was accompanied by growth inhibition and sharp reduction of UDPG pool. It would be reasonable to suppose a competition between ADPG and UDPG biosyntheses because some precursors are common for both of them. UDPG is most intensively used by plant cells in many transglycosyla­tion reactions (Turner and Turner, 1975) some of which appear to be essential for cell growth. The inhibition of UDPG biosynthesis may consequently be one of the rea­son of adenine-induced growth retardation.

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