Stimulation of DNA synthesis and DNA polymerase activity by benzyladenine during early germination of maize axes

JORGE M. VAZQIJEZ-RAMOS AND JORGE REYES JIMENEZ Departamento de Bioquimica, Divisi6n de Bioquimica y Farmacia, Facultad de Quimica, Ciudad Universitaria. Avenida

Insurgentes Sur y Copilco, 04510 MPxico, D. F.

Received February 5, 1990

VAZQUEZ-RAMOS, J. M., and JIMENEZ, J. R. 1990. Stimulation of DNA synthesis and DNA polymerase activity by benzyladenine during early germination of maize axes. Can. J. Bot. 68: 2590-2594.

Benzyladenine, a synthetic cytokinin, stimulates DNA synthesis during early germination of maize. A peak of stimulation is observed at 6 h in the period of 0-9 h of imbibition of embryo axes. Stimulation of DNA synthesis by the phytoregulator is dependent on RNA and (or) protein synthesis, since a-amanitin and cycloheximide inhibit such stimulation. Although DNA polymerase activity is stimulated by incubation of axes in the presence of benzyladenine, enzyme amount is apparently not increased. An enzyme located in the nuclei seems to be preferentially stimulated, and studies with specific inhibitors for polymerases and use of different templates suggest this enzyme could be of type y. The nature of the mechanism of stimulation of DNA synthesis is discussed.

Key words: benzyladenine, DNA synthesis, DNA polymerase, maize, germination.

VAZQUEZ-RAMOS, J. M., et JIMENEZ, J. R. 1990. Stimulation of DNA synthesis and DNA polymerase activity by benzyladenine during early germination of maize axes. Can. J. Bot. 68 : 2590-2594.

La benzyladenine, une cytokinine synthktique, stimule la synthbse de I'ADN au debut de la germination chez le mais. Au cours d'une periode d'imbibition des axes embryonnaires de 0-9 h, on observe un maximum de stimulation i 6 h. La stimulation de la synthbse d'ADN par le phytorCgulateur depend de la synthbse de I'ARN et (ou) des protCines, puisque l'a-manitine et la cycloheximide prkviennent cette stimulation. Bien que l'activitk de I'ADN polymerase soit stimulCe par I'incubation d'axes en prksence de benzyladenine, la quantitC d'enzyme ne semble pas cependant augmenter. I1 semble qu'une enzyme situCe dans les noyaux serait prCfLrentiellement stimulCe; des Ctudes conduites avec des inhibiteurs spkcifiques pour les polymCrases et differentes mesures de rCfCrences suggkrent que cette enzyme serait de type y. Les auteurs discutent la nature du mCcanisme de stimulation de la synthbse d'ADN.

Mots clPs : benzyladbnine, synthbse d'ADN, ADN polymbrase, mays, germination. [Traduit par la revue]

Introduction albumin, calf thymus DNA, dATP, dCTP, dGTP, ATP, poly rA

After entry of water into cereal seeds, a complex series of metabolic events is triggered which will lead the seeds to ger- minate and finally grow. Chemical messengers such as the phytoregulators or phytohormones seem to play a very impor- tant role during both germination and further development of the plant (22).

Cytokinins, one of the five classes of phytoregulators known, have been found to participate in several metabolic events of plant development: they increase the rate of chlo- roplast biogenesis and of synthesis of ribulose bisphosphate carboxylase (1 3, 15), delay senescence (3, 21), stimulate nucleic acid synthesis and cell division (8, 16, 17, 27, 29), and promote differentiation (4, 20). The mechanism of action of cytokinins is still largely unknown.

In recent years, cytokinins have been found to stimulate seed germination (23), promoting early DNA synthesis in cotyle- dons of watermelon seeds (9), and increasing total DNA syn- thesis and enhancing repair-type DNA synthesis in maize embryo axes (28). Benzyladenine (BA), a synthetic cytokinin, stimulated repair-type DNA synthesis in y-irradiated axes and helped define the nature of the early DNA synthesis as repair- type in nonirradiated axes (28).

In this paper, an attempt is made to understand at which level BA is stimulating early DNA synthesis in maize axes.

Materials and methods Chemicals

Benzyladenine, phenylmethylsulfonyl fluoride (PMSF), chloram- phenicol, a-amanitin, cycloheximide, thymidine, bovine serum

(Sigma P9403), -oligo dT (5), aphidicolin, N-ethylmaleimihe,- and dideoxythymidine triphosphate (ddTTP) were purchased from Sigma Chemical Co., St. Louis, MO. Tris(hydroxymethy1) aminometh- ane(Tris), 2,5-diphenyloxazole (PPO), and 2,2'-p-phenylene- bis[5-phenyloxazole] (POPOP) were from Merck, MCxico. [MethylL3H]thymidine (20 Ci/mmol) (1 Ci = 37 GBq) and [methyl- 3H]thymidine 5'-triphosphate (TTP, 75 Ci/mmol) were from New England Nuclear.

Plant material Zea mays var. Chalqueiio was kindly provided by Productora

Nacional de Semillas (Pronase), Coyoachn, MCxico, D.F. It had a percentage germination of 95% after 48 h. Maize embryo axes were dissected from the seed by hand and used within the next 24 h.

Irradiation of maize embryo axes Embryo axes in plastic Petri dishes were exposed to y irradiation

from a cobalt source in a Gamma Cell 200 (AECL) irradiator for a period of 120 min to achieve an irradiation dose of 1000 Gy.

Imbibition of maize embryo axes Maize axes (10 per sample) were surface sterilized with 0.5%

NaClO (vlv, 2 mL)) by shaking for 1 min and then washing four times with 4-mL volumes of sterile distilled water. Excess viater was removed by blotting the axes against sterile Whatman paper NO. 1. 'They were then incubated at 27OC between two discs of sterile Whatman paper No. 1 with sterile imbibition buffer consisting of 50 mM KCI, 10 mM MgCl,, 50 mM Tris-HCI pH 7.6, 2% sucrose, and chloramphenicol (10 p,g/mL): [Methyl-3H]thymidine (10 pCi/mL) was added when thymidine incorporation into TCA- insoluble material was to be followed. Specific conditions of labelling

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for each experiment are indicated in the text. BA was added, when needed, at a concentration of lo-'' M.

When u-amanitin (1.1 x lo-' M) or cycloheximide (3.6 x M) were used, they were introduced to the axes under vacuum

conditions, i.e., the inhibitors were added at the required concentra- tions to the imbibition buffer with or without BA and the complete mixture was added to the axes and these subjected to vacuum con- ditions for 10 min. The axes were then incubated as described above, without further addition of buffer. Control samples (no inhibitor) were also vacuum treated with imbibition buffer as described above.

["H]Thymiditle incorporatiorz After incubation, the axes were washed once with I % sodium cit-

rate (w/v, 10 mL) and once with 80% ethanol (vlv, 10 mL), both containing nonradioactive thymidine (200 pg/mL). The axes, if not processed immediately, were frozen at - 70°C until use.

The frozen samples were thawed and 80% ethanol (v/v, 1.5 mL) containing nonradioactive thymidine (200 pg/mL) was added. The samples were homogenized at 4OC in a Polytron PCU-2 homogenizer at full speed and then spun for 10 min at 2600 x g. The supernatant was discarded and the pellet resuspended in I M NaOH (0.5 mL), heated at 90°C for 3 min, and then cooled. TCA (10% w/v. 2 mL) was then added, and the samples were maintained on ice for at least 30 min, then vacuum filtered through Whatman GF/A glass fiber filters (2.4 cm), washed with 570 TCA (w/v, 5 mL) and 95% ethanol (10 mL), dried, and counted in a Packard Tri-Carb Scintillation Counter.

Isolation of DNA polymerase (24) Embryo axes (150 mg) were surface sterilized and imbibed as

before. The following manipulations were carried out at 4OC: axes were homogenized with 3 mL of buffer A (50 mM Tris-HC1 pH 7.6, 25 mM KCI, 1 mM 2-mercaptoethanol, 0.25 M sucrose, and 0.1 mM PMSF) in a Polytron PCU-2 homogenizer at full speed. The homog- enate was centrifuged twice at 5000 X g for 15 min each time, and the supernatant was then centrifuged at 100 000 X g for 2 h. The resulting supernatant was used as the total enzyme source.

Isolation of nuclei (6) and nuclear DNA polymerase Embryo axes (1 g) were homogenized in a mortar at 4OC with 2 mL

of buffer B (50 mM Tris-HCI pH 7.5 , 20 mM KCl, 20 mM MgCl,, 10 mM 2-mercaptoethanol, 1.2 M sucrose, and 30% glycerol). The homogenate was vacuum filtered through two layers of Miracloth and washed twice with 4-mL volumes of the same buffer. The filtrate was centrifuged 40 min at 20 000 X g . The supernatant was dialyzed against dialysis buffer (see below) and was used as the soluble enzyme source. The pellet, which contained nuclei, was washed once with buffer B but containing 0.3 M sucrose and then resuspended in 1.5 mL of buffer C (50 mM Tris-HCI pH 7.5, 5 mM MgCl?, 0.5 mM 2-mercaptoethanol, 0.1 mM PMSF, 0 . I mM EDTA, and 5% glyc- erol). The suspension was frozen (-70°C) and thawed three times and then centrifuged 2 h at I00 000 x g. The resulting nuclear super- natant was the source of enzyme. Minimal DNA polymerase activity was detected in the residual pellet. The nuclear supernatant was dialyzed overnight against dialysis buffer (buffer C without EDTA).

DNA polymerase assay DNA polymerase activity was assayed in 100 p L of the following

mixture (25): 25 mM Tris-HCI pH 7.6, 16 mM KCI, 6 mM MgCI?, 0.1 mM each of dATP, dGTP, and dCTP, 1 mM ATP, 4% glycerol, 0 .4 mM 2-mercaptoethanol, 5 p g of activated calf thymus DNA, 20 p L of crude enzyme preparation, and 2 pCi [methylL3H] TTP. Incubation was canied out at 37OC for 30 min. Activated calf thymus DNA was prepared by limited digestion of calf thymus DNA with pancreatic DNase as described in ( I ) . The reaction was stopped by the addition of 100 pL of a solution of 2 mg salmon sperm DNAImL and 2 mL of 1070 cold TCA. These samples were placed on ice for 30-60 min and the TCA-insoluble material was measured in a Packard Tri-Carb Scintillation Counter. When DNA polymerase

TABLE 1. Stimulation of DNA synthesis during early germination by BA (n = 4)

--

BA-treated axes: % increase ( 2 SD) over nontreated axes

Imbibition time (h) Nonirradiated axes y-Irradiated axes

3 1 9 i 9 21 2 2 6 125 i 15 7 2 2 2 1 9 5 7 2 12 4 8 2 17

12 151 t_40 7 4 k 9

NOTE: Average c p d l O embryo axes for nonirradiated axes. with and without BA, respectively: 1865 and 2220 after 3 h. 2060 and 3642 after 6 h, 2980 and 4679 afler 9 h. and 6286 and 15 778 after I2 h. and for y-irradiated axes, 2298 and 2781 after 3 h. -1253 and 7403 afrer 6 h. 2186 and 3235 afrcr 9 h. and 3714 and 6462 after I2 h.

inhibitors were used, their concentrations were 1.5 x l o - ' M aphi- dicolin and 1.0 x lo- ' M N-ethylmaleimide (both dissolved in DMSO), and 3.1 x lo- ' M ddTTP (dissolved in water). Controls for aphidicolin and N-ethylmaleimide received the corresponding concentration of DMSO.

Use of poly rA - oligo dT The synthetic polynucleotide and oligodeoxynucleotide were dis-

solved separately in buffer containing 10 mM Tris-HCI pH 7.5. 500 p M MnCl,, and 0.5 mg BSAImL, at a concentration of 0.5 and 0.084 pgIpL, respectively. For the reaction. the conditions were as described for the DNA polymerase assay except that the activated DNA was substituted by a mixture of 10 p L each of poly rA and oligo dT, giving a template to primer ratio of 10: 1, and 50 mM KPO, was added to inhibit P-polymerase activity. The method was adapted from Knopf (12) and Vishwanatha (26).

Proreit1 determination Protein concentration was determined by the method of Lowry, as

modified by Peterson (1 8).

Results

Differential stitni~lation of DNA sytlrhpsis during early ger- rnirzatiotz by BA

Previous findings indicated that BA stimulated DNA synthesis during early germination (0-3 h) in both y- and non- irradiated maize axes (28). We followed the pattern of stim- ulation in these two tissues throughout the first 12 h of ger- mination, with BA present all the time. ['HIThynlidine was added in 3-h pulses during the last 3 h of imbibition. Table 1 shows that there is stimulation at all times tested, but maximal stimulation was achieved at 6 and 12 h in both types of tissues. The peak at 12 h, however, might represent DNA replication already taking place, at least in nonirradiated axes (2). If ['Hlthymidine incorporation is followed (note to Table l ) , it can be observed that at 6 h, y-irradiated axes accumulate more label (4255 and 7403 cpm for - BA and + BA, respectively) than nonirradiated axes (2060 and 4642 cpm for -BA and + BA, respectively; see also note to Table 2); there is a drop of incorporation by 9 h in both tissues, and thereafter DNA synthesis in nonirradiated axes is greater than that of y-irra- diated axes.

Effect of cycloheximide atzd a-atnanitin on stit?z~~latiotz of DNA synthesis by BA at 6 h of maize axes imbibitiorz

To understand the nature of the mechanism of stimulation of DNA synthesis by BA, inhibitors of RNA synthesis (a- amanitin) and protein synthesis (cycloheximide) were used; the time chosen for these experiments was 6 h, since at this time maximal stimulation was observed (see Table 1). Cyclohexi-

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2592 CAN. J . BOT. VOL. 68. 1990

TABLE 2. Effect of cycloheximide (CH) and a-amanitin (AA) on DNA TABLE 4. Effect of cycloheximide and a-amanitin on synthesis in axes after 6 h of imbibition (n = 3) specific activity (cpmlpg protein) of total DNA poly-

merase extracted from treated axes imbibed for 6 h Imbibition Percentage of control ( % SD) (n = 3) conditions Nonirradiated axes y-Irradiated axes

- BA 100 100 -BA +CH 5 4 2 3 52? 6 -BA +AA 5 0 % 4 65 2 6 + BA 146 % 3 142%3 +BA +CH 60? 6 6 3 5 +BA +AA 64? 2 68?4

NOTE: Average c p d l O embryo axes for nonirradiated and y-irradiated axes, respec- tively: - BA, 8803 and 26 174; - BA +CH, 8784 and 13 610; - BA + AA, 5209 and 8504; + BA, 12 877 and 37 700; + BA +CH, 9757 and 16 386; + BA + AA, 6747 and 8912.

TABLE 3. Specific activity ( c p d p g protein) of total DNA polymerase extracted from axes imbibed for 6 h

Imbibition Specific activity Percentage conditions (%SD) of control

Nonirradiated - BA 1 2 0 5 7 100 + BA 144 2 5 120

y-Irradiated - BA 111?4 100 + BA 128%7 115

mide not only blocked the stimulatory effect caused by BA but also reduced DNA synthesis to approximately 60% of that in nonstimulated axes in both nonirradiated and irradiated axes (Table 2). A similar reduction was observed when cyclohex- imide was added to axes (irradiated or not) imbibed in the absence of BA. a-Amanitin also blocked the effect of BA and reduced DNA synthesis to approximately 65% of that in non- stimulated axes (irradiated or not) (Table 2). Thus, it appeared that stimulation of transcription and (or) translation was part of the mechanism by which BA stimulated DNA synthesis and that this mechanism was similar for both types of tissues. In the above experiments (Table 2), it can be seen that stimulation of DNA synthesis by BA was not as high as that shown in Table 1 . A possible explanation for this discrepancy may be associated with the experimental conditions; because both inhibitors were introduced into axes under vacuum (see Mate- rials and methods), the control cells in Table 2 were also vac- uum treated, but not the cells in Table 1. It is also important to indicate that under the experimental conditions cyclohexi- mide inhibited protein synthesis (as measured by incorporation of [35S]methionine into TCA-insoluble material) more than 95% and a-amanitin, about 30%. The explanation for the small inhibition by a-amanitin is that most protein synthesis during early germination comes from stored mRNA (19).

Effect of BA on DNA polymerase activity Stimulation of DNA synthesis by BA could be due to stim-

ulation of DNA polymerase activity. Therefore, this enzyme was measured as previously reported (24) in extracts of both y- and non-irradiated axes after a 6-h imbibition period. Incu- bation of axes with BA produced an increase in DNA poly- merase activity of 15 and 20% in y- and non-irradiated axes, respectively (Table 3).

Effect of a-amanitin and cycloheximide on the stimulatory effect of BA on DNA polymerase activity

The higher polymerase activity in BA-treated axes could be due to an increase in the amount of DNA polymerase. To test

Imbibition Specific activity Percentage conditions (? SD) of control

TABLE 5. Nuclear and soluble DNA polymerase specific activity ( c p d p g protein) from BA-stimulated axes imbibed for 6 h and the

effect of inhibitors (n = 3)

Assay Specific activity Enzyme conditions ( % SD) %

Soluble No addition 6 3 % 7 100 + BA 63 % 9 100

Nuclear No addition 333% 17 100 + BA 390?26 117 + aphi 253 % 24 76 +aphi +BA 233 ? 20 70 + ddTrP 3 0 2 1 1 9 + d d m P + BA 1 7 2 2 5 + NEM 67?9 20 +NEM +BA 6 7 2 1 1 20

NOTE: aphi, aphidicolin; ddTTP, dideoxythymidine lriphosphate; NEM, N-ethylmaleimide.

this possibility, axes were incubated for 6 h with or without BA in the presence of either a-amanitin or cycloheximide. In the presence of BA, DNA polymerase activity was 19% higher than in control axes (Table 4). However, when cycloheximide or a-amanitin was present in axes with or without BA, DNA polymerase activity remained equal to or increased above the level found in nonstimulated control axes (Table 4). Similar results were observed for y-irradiated axes (not shown).

Localization and nature of the stimulated DNA polymerase We also measured DNA polymerase activity in both the

nuclear supernatants and the supernatants of the nuclei isola- tion (soluble enzyme) that originated from either control (no BA) or BA-treated axes incubated for 6 h. Table 5 shows that there is an increase of 17% in nuclear DNA polymerase activ- ity when axes are incubated in the presence of BA, whereas there is no increase in soluble DNA polymerase activity. The nuclear activity was further characterized by means of inhib- itors. It was only moderately inhibited by aphidicolin (inhibitor of a-type polymerases) whether axes were incubated with BA or not (24 and 30%, respectively), whereas both N-ethylmal- eimide (inhibitor of a and y type polymerases) and ddTTP (inhibitor of P and y polymerases) almost totally inhibited enzyme activity from both stimulated and nonstimulated axes. Additionally, the nuclear enzyme could use the synthetic tem- plate poly rA - oligo dT with 55% (BA-stimulated axes) and 52% (nonstimulated axes) efficiency with respect to activated DNA (Table 6).

Discussion Stimulation of DNA synthesis during early seed germination

by BA seems to be a common phenomenon (9,28). Of interest

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TABLE 6. Use of natural and synthetic templates by nuclear DNA polymerase

Assay Specific activity Efficiency conditions Temolate ( C D ~ L L P orotein) of use (%)

- BA Activated DNA 476 100 poly rA - oligo dT 248 52

+ BA Activated DNA 540 100 poly rA - oligo dT 298 55

N u r ~ i : Average of two experiments

are the findings that the phytoregulator greatly enhances repair- type DNA synthesis in y-irradiated axes and that it changes the pattern of synthesis from an undefined type to a repair-type synthesis (as defined by BND-cellulose chromatography) in nonirradiated axes (28). The mechanism of stimulation is unknown.

As a first approach to understand at what level BA is pro- moting its effect, protein and RNA snythesis inhibitors were used in both y- and non-irradiated axes. The inhibitors reduced DNA synthesis by the same amount in both types of tissues (Table 2), suggesting that the mechanism of stimulation of early DNA synthesis and DNA polymerase activity by BA seems to be the same in both y - and non-irradiated axes (at least up to 6 h), and that no difference exists at the biochemical level. O n the other hand, these results indicate that early DNA synthesis depends o n the production of new proteins necessary for DNA metabolism, and also that BA-dependent DNA syn- thesis is a consequence of stimulation at the level of transcrip- tion-translation.

W e believed that stimulation of DNA polymerase by BA during early germination could explain the results reported above. W e found that DNA polymerase activity was enhanced by the presence of BA (Table 3). However, this increase in activity is not paralleled by an increase in the protein itself, as suggested by the experiments with RNA and protein synthesis inhibitors (Table 4). Since in vivo experiments show that DNA synthesis is highly reduced by the inhibitors while DNA poly- merase activity does not seem to change, two conclusions can then be drawn: (i) since DNA polymerase activity is not affected by the presence of any of the inhibitors during axes incubation, it is not a decrease in this enzyme that causes the decrease of DNA synthesis when axes are imbibed in the pres- ence of a-amanitin or cycloheximide (Table 2), and (ii) DNA polymerase does not seem to be synthesized during early ger- mination, a finding already reported elsewhere (25).

Therefore, the increase in DNA polymerase activity caused by BA could be the result of either the modification of the enzyme itself via the stimulation by BA of the synthesis of a factor that would modify DNA polymerase or the synthesis of any other factor necessary for DNA synthesis. Supporting the former is the finding that phosphorylation of rat fibroblast DNA polymerase a and Xetlopus DNA topoisomerase I changes their catalytic activities (7, 11). Variations in the level of modifi- cation of DNA polymerase (phosphorylation-dephosphoryla- tion, etc.) might explain the results reported in this paper. Experiments in this context are already in progress.

Interestingly, it is mainly the nuclear enzyme that is stim- ulated by BA. Considering its response to inhibitors, the nuclear enzyme appears to be more a y-type enzyme: N-ethyl- maleimide and ddTTP have a strong inhibitory effect and both are inhibitors of mammalian y activities (14), whereas a activities are very insensitive to ddTTP and P activities should

be insensitive to N-ethylmaleimide. Moreover, this enzyme uses with good efficiency a template used only by mammalian y-enzymes, i .e. , poly rA - oligo dT. This rules out the pos- sibility that the enzyme is P-type because this substrate is not used by P enzymes (5); y-type enzymes have also been reported to be present in nuclei of germinating wheat embryos (10). W e are currently trying to characterize this enzyme further.

Acknowledgment

This research was supported by Consejo Nacional d e Cien- cia y Technologia (CONACYT) grant No. 880374.

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