Planta (Berl.) 127, 63--68 (1975) 9 by Springer-Verlag 1975

Onset of Nucleic Acid Synthesis during Germination of Pisum sativum L. Nigel E. Robinson* and John A. Bryant** Nottingham University School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, U.K. Received 25 June; accepted 28 July 1975

Summary. Measurements of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximately 30 h after the onset of germination. The onset of net nucleic acid synthesis was marked by an increase in the rate of incorporation of [aH]thymidine into DNA, and of [att]orotie acid and [aH]uridine into both DNA and RNA. rRNA was usually more heavily labelled than tRNA, but was not preferentially accumulated, suggesting a grater rate of turnover of rRNA than tRNA. Some incorporation of precursors occurred prior to ~he onset of net nucleic acid synthesis, particularly into RNA. This was taken to represent nucleic acid turnover. There was no evidence that the "scavenging" pathways for nucleotide biosynthesis were more important than the "normal" pathways in contributing precursors for net nucleic acid synthesis. Introduction

The onset of nucleic acid synthesis in the embryonic axis during germination has been studied in a number of plants. The results of these studies have varied considerably according to the species of plant under investigation. However, one feature is common to all the species studied, namely that there is a sig~aificant lag phase between the onset of germination and the onset of net synthesis of DNA and RNA. I n several plants, including Vicia and Gossypium, net synthesis of DNA and net synthesis of RIffA are initiated at the same time (Jakob and Bovey, 1969; Clay et al., 1975) whereas in other plants, e.g. Allium, net synthesis of R N A precedes net synthesis of DNA (Melera, 1971). I n a number of species, cells in the embryonic axis are able to incorporate isotopically-labelled precursors into RIgA prior to the onset of measureable net RNA synthesis. Some investigators have suggested that this incorporation represents synthesis o f m R N A (Van de Walle, et at., 1973), but Walbot (i972) has reported that in Phaseolus, 1971 ; Dobrzs the incorporation of precursors into RNA, which occurs prior to the onset of massive net R N A synthesis, represents synthesis of rRNA and tRNA. I t has also been reported that, following the onset of net RNA synthesis in Raphanus, Triticum and Goasypium, rRNA is synthesised at a far greater rate than tl~NA (Julien et al., 1970; Chen et al., 1971 ; I)obrzs et al., 1973; Clay et al., 1975). However,

* Present address: Department of Botany, School of Biological Sciences, University of Leicester, Leicester, U.K. ** Present address: Department of Botany, University College, Cardiff CF1 1XL, U.K.

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N . E . Robinson and J. A. Bryant

in A l l i u m a n d Phaseolus, r R N A a n d t R ~ A are synthesised a t t h e same r a t e (Melera, 1971; W a l b o t , 1972). W e are i n v e s t i g a t i n g t h e control of nucleic acid biosynthesis during germinat i o n of P i s u m sativum. Because of the g r e a t interspecific v a r i a t i o n w t i h r e g a r d to t h e onset of nucleic acid synthesis, it is clearly necessary to characterize t h e p a t t e r n s of nulceic acid biosynthesis in P . sativum prior to a n y i n v e s t i g a t i o n of control mechanisms. I n this p a p e r we r e p o r t results f r o m e s t i m a t i o n of t o t a l nucleic acid c o n t e n t a n d f r o m studies of i n c o r p o r a t i o n of radioisotopieally labelled precursors i n t o t h e nucleic acids of t h e e m b r y o n i c axes of g e r m i n a t i n g peas.

Materials and Methods

Growth el Plants. For experiments involving the use of radioisotopes, pea seeds (Pisum sativum L., c.v. Feltham :First) were surface-sterilised in 2 % (v/v) peracetic acid containing 0.16% (w/v) sodium alkyl-aryl-sulphonate. The seeds were washed and then soaked for 4 hrs in sterile distilled water, and planted in autoclaved vermiculite moistened with sterile distilled water. The seeds were allowed to germinate under aseptic conditions at 22 ~ C in the

light. For all other experiments, seeds were surface-sterilised in sodium hypochlorite (2.5%, w/v, available chlorine), washed for 4 hrs in running tap water, and then planted in moist vermiculite. The seeds were allowed to germinate at 22 ~ C, in the light. In both types of experiment, the beginning of the period of surface-sterilisation was taken as the onset of the germination process (i.e. time zero). Estimation of Total Nucleic Acids. At intervals after the onset of germination, samples of seeds (10 seeds per sample) were taken. The embryonic axes were dissected from the seeds and homogenised in absolute ethanol at room temperature. Nucleic acids were extracted from the homogcnates essentially as described by Guinn (1966). The nucleic acid solutions were scanned in the ultraviolet region of the spectrum. The absorption spectra were characteristic of nucleic acids, with maxima at 260 nm and minima at 230 nm. The total nucleic acid content was determined from the absorption at 260 nm, correction being made for the absorption due to proteins by determination of the 260 nm:280 nm absorption ratios. DNA was estimated by the diphenylamine reaction (Burton, 1956). The extracts contained no arabinose or uronie acids, both of which interfere with the diphcnylamine reaction (Bryant and ap Rees, 1971). Labelling and Extraction el Undegraded Nucleic Acids. At intervals after the onset of germination, samples of aseptically grown seeds were transferred under aspetic conditions to petri dishes containing 15 ml of a solution of chloramphenieol and streptomycin sulphate (each at 25 tzg/ml), plus one of the following compounds: 200 aCi [Me-aH]thymidine (40 Ci/ retool) or 500 fzCi [5-all]erotic acid (25 Ci/mmol) or 250 ~zCi [5,6-aH]uridine (50 Ci/mmol). After 4 hrs incubation the seeds were washed in distilled water. The embryonic axes were excised and used for extraction of tmdegraded nucleic acid. For studies of incorporation of precursors into DNA, embryonic axes were homogenised in 4 % (w/v) sodium lanryl sulphate. DNA was extracted from the homogenate as described earlier (Bryant and Wildon, 1971). For studies of incorporation of precursors into P~NA, embryonic axes were homogenised in a solution containing 6% (w/v) 4-amino salicylic acid (sodium salt), 5% (w/v) phenol, 1% (w/v) tri-iso.propylnaphthalene sulphonic acid (sodium salt) and 1% (w/v) NaC1. Undegraded nucleic acids were extracted from the homogenate as described by Loening and Ingle (1967). Fractionation o/Nucleic Acids. Undegraded DNA was fractionated on columns of poly-Llysine kieselguhr, as described by Bourne et al. (1974). Fractions were assayed for absorbance at 260 nm and for radio-activity as described previously (Bourne et al., 1974). Total undegraded nucleic acids were fractionated by electrophoresis in 2.4% (w/v) polyacrylamide gels, containing 0.6% (w/v) bis-acrylamide (Loening, 1967) using a current of 5 mA/gel for 2 h. The gels were scanned at 265 nm in order to locate and estimate the nucleic acids, and then frozen and cut into slices of 1 mm thickness. Radioactivity in the slices was assayed by a method similar to that described by Grierson and Smith (1973).

Nucleic Acid Synthesis during Germination

65

Results and Discussion

Nucleic Acid Content of Embryonic Axis Fig. 1 clearly shows that there was little or no net nucleic acid synthesis in the embryonic axis during the first 30 h of germination. From 30 h onwards, high rates of R N A and DNA accumulation were observed. There was no evidence for the synchronous waves of nucleic acid synthesis which have been reported to occur in Fieia (Jakob and Bovey, 1969). The onset of net nucleic acid synthesis in the embryonic axis coincided with the dramatic increase in the fresh weight of the axis.

Incorporation o] Labelled Precursors Into the Nucleic Acids o] the Embryonic Axis The rate of incorporation of radioactivity from [3H]thymidine into DNA very clearly paralleled the rate of net DNA synthesis (Table 1). There was very little incorporation up to 25 h after the onset of germination. The rate of incorporation at 29-33 h was 40 times that at 21-25 h, and a further five-fold increase in rate occurred between 33 h and 45 h. Orotic acid and uridine were much less efficiently used than thymidine for DNA synthesis (Tables 2 and 3). Neither did their rates of incorporation into DNA reflect the rate of net accumulation of DNA as well as did the rate of thymidine incorporation. However, there was a 6- to 7-fold increase in the rate of incorporation of radioactivity from [3H]orotic acid into DNA and an 8-fold increase in the rate of incorporation of radioactivity from [3H]uridine into DNA, following the onset of net DNA synthesis. The low levels of incorporation of radioactivity from thymidine, orotic acid and uridine observed prior to the onset of net DNA synthesis may represent DNA turnover (Bryant, Wildon and Wong, 1974) or the synthesis of a minor fraction of DNA, such as mitochondrial DNA.

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Fig. 1. Changes in the total nucleic acid content and in the fresh weight of the embryonic axis during germination of Pisum sativum. Each point represents the mean of 4 or 6 determinations 5

Plan~a (Berl.)

66

N . E . Robinson and J. A. Bryant

Table 1. Incorporation of radioactivity from [Me-3H]thymidine into the DNA of the embryonic axis during germination Pea seeds were germinated and grown aseptically. At intervals samples of seeds were supplied with [Me-aH]thymidine for 4 h. The DNA was extracted from the embryonic axes and fractionated by chromatography on columns of poly-L-lysine kieselguhr. Column fractions were assayed for absorbance at 260 nm and for radioaeitivity Time (h)

pmol radioactive nucleoside incorporated] ~mol supplied]rag DNA/4 h

4- 8 21-25 29-33 45-49

134 134 5180 26 500

Table 2. Incorporation of radiactivity from [5-3H]orotie acid into nucleic acids of the embryonic acis during germination Pea seeds were germinated and grown aseptically. At intervals, samples of seeds were supplied with [5-3H]orotic acid for 4 h. Nucleic acids were extracted from the embryonic axes and ffactionated by polyaerylamide gel electrophoresis. The gels were scanned at 265 nm in order to locate and estimate the nucleic acids, and then sliced and counted Time (h)

pmol radioactive base incorporated] ~mol supplied/rag nucleic acid/4 h DNA

4- 8 21-25 29-33 45-49

5.1 15.1 34.8 30.9

25s and 18s rRNA 21.0 30.2 90.2 246

5s and 4s RNA 15.4 19.7 148 217

Table 3. Incorporation of radioactivity from [5,6-3H]uridine into nucleic acids of the embryonic axis during germination The experiments were carried out as described in the caption of Table 2, except that [5,6-3H]uridine was used instead of [5-3H]orotic acid Time (h)

4- 8 21-25 29-33 45-49

pmol radioactive nucleoside incorporated] ~mol supplied]rag nucleic acid/4 h DNA

25s and 18s rRNA

5s and 4s RNA

16.8 24.0 142 193

104 74.2 170 361

55.6 63.2 127 207

T h e d a t a relating to i n c o r p o r a t i o n of r a d i o a c t i v i t y into R N A show 4 m a j o r features (Tables 2 a n d 3). Firstly, r a d i o a c t i v i t y from erotic acid a n d uridine was i n c o r p o r a t e d i n t o R N A i n significant a m o u n t s d u r i n g the period before t h e onset of n e t RI~A synthesis. W e t a k e this i n c o r p o r a t i o n to represent R N A t u r n o v e r

Nucleic Acid Synthesis during Germination

67

(Trewavas, 1970), although it is also possible t h a t a low rate of net synthesis, not readily detectable b y estimation of total RNA content, occurred prior to the onset of massive net R N A synthesis. Secondly, orotic acid and uridine were not as efficiently used as precursors for R N A synthesis as was thymidine for DNA synthesis. The relatively low level of incorporation of radioactivity from orotic acid and uridine into RNA and DNA (compared with the incorporation of radioactivity from thymidine into DNA) suggests t h a t in the embryonic axis of pea, as in other plant cells, the pool of ribonucleotides is much larger t h a n the pool of deoxyribonueleotides (Nygaard, 1972). Thirdly, in the period prior to the onset of massive net RNA synthesis, uridine was used much more efficiently t h a n orotic acid as a precursor for R N A synthesis. After the onset of net RNA synthesis, there was little difference in the utilisatiou of the two compounds. The activity of uridine kinase is known to be high during the first 30 h of germination (J. A. Bryant, submitted for publication), and it seems likely t h a t during the lag phase before the onset of net nucleic acid synthesis, the " s c a v e n g i n g ' p a t h w a y for the synthesis of pyrimidine nucleotides is more active t h a n the "normal" pathway. Fourthly, the rate of incorporation of radioactivity from orotic acid or uridine into r R N A was nearly always somewhat greater than the rate of incorporation into tRNA. Since the U.V. scans of the polyacrylamide gels gave no evidence for a selective accumulation of r R N A during germination, the greater rate of labelling of r R N A probably represents a greater rate of turnover of r R N A as compared with t R N A (c.f. Trewavas, 1970). The results obtained from studies of the utilisation of radioactive precursors thus provide evidence for an increase in the rate of incorporation into both D N A and RNA at approximately 30 h after the onset of germination. The increased rate of incorporation cannot be explained in terms of an increased rate of uptake, since estimates of radioactivty in the soluble phase of cell homogenates indicated t h a t the uptake of radioactivity per unit fresh weight at 29 to 33 h was only 1.2 to 1.7 times t h a t at 4 to 8 h. The increase in incorporation rate was nearly always much greater than 1.7-fold. Neither was increased incorporation of radioactivity from uridine and thymidine caused by increase in the activities of uridine and thymidine kinases. The activities of these enzymes in the embryonic acis are high, even in the very early stages of germination, and further, do not show marked increaeses until several hours after the increases in incorporation rates (J. A. Bryant, submitted for publication). Thus, the changes in incorporation rates observed in our experiments m a y be taken to represent changes in the rates of synthesis of the nucleic acids. Further, after making allowance for the occurrence of R N A turnover, and, to a much lesser extent, of DNA turnover, the changes in rates of incorporation of radioactivity also reflect the changes in the rates of net accumulation of the nucleic acids. The pattern of onset of nucleic acid synthesis described here is similar to t h a t described for other leguminous plants. I n particular, the marked simultaneous increases in the rates of synthesis of DNA and RNA have also been described in Vicia (Jakob and Bovey, 1969). Further, the phase prior to the onset of net DNA synthesis which we observed in seedlings of Pisum grown at 22 o C is comparable in length to t h a t observed b y Davidson (1966) in Vicia seedlings grown at 20 ~ C. (30 h in Pisum; 32 h in Vicia). Lastly, the observations t h a t cells in the embryonic

68

N . E . Robinson and J. A. Bryant

axis of P i s u m are able to i n c o r p o r a t e r a d i o a c t i v i t y from labelled precursors i n t o R N A p r i o r to t h e onset of r a p i d n e t s y n t h e s i s of R N A a r e v e r y similar t o observations m a d e on Phaseolus e m b r y o s ( W a l b o t , 1972). F r o m our m e a s u r e m e n t s of t h e r e l a t i v e r a t e s of s y n t h e s i s of r R N A , t R N A a n d D57A, we can find no evidence for t h e " c a s c a d e a c t i v a t i o n " of genome transcription described by Dobrs et al. (1973). H o w e v e r , i t m u s t be p o i n t e d o u t t h a t our m e t h o d s d i d n o t p e r m i t s t u d y of m R N A m e t a b o l i s m , a n d i t r e m a i n s possible t h a t m l ~ N A b i o s y n t h e s i s is i n i t i a t e d earlier in g e r m i n a t i o n t h a n t h e bios y n t h e s i s of o t h e r nucleic a c i d species. This p r o b l e m is c u r r e n t l y u n d e r investigation, N.E.R. is grateful to the Ministry of Agriculture, Fisheries and Food for financial support. References Bourne, R.A., Bryant, J. A., Falconer, I. R.: Stimulation of DNA synthesis by prolactin in rabbit mammary tissue. J. Cell Sci. 14, 105-111 (1974) Bryant, J. A., ap Rees, T. : Nucleic acid synthesis and induced respiration by disks of carrot storage tissue. Phytochem. 19, 1191-1197 (1971) Bryant, J. A., Wildon, D. C. : Criticism of the use of methylated albumin-kieselguhr chromatography for the isolation of metabolically labile DNA. Bioehim. biophys. Aeta (Amst.) 282, 624-629 (1971) Bryant, J . A . , Witdon, D.C., Wong, D.: Metabolically labile DNA in aseptically grown seedlings of Pisum 8ativum L. Planta (Berl.) 118, 17-24 (1974) Burton, K. : A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323 (1956) Chen, D., Sehultz, G., Katehalski, E. : Early ribosomal RNA transcription and apperanee of cytoplasmic ribsomes during germination of wheat embryo. Nature (Lond). New Biol. 281, 69-72 (1971) Clay, W. F., Katterman, F. R. H., Hammett, J. iR.: Nucleic acid metabolism during germination of Pima cotton (Gossypium barbadense). Plant Physiol. 55, 231-236 (1975) Davidson, D.: The onset of mitosis and DNA synthesis in roots of germinating beans. Amer. J. Bot. 53, 491-495 (1966) Dobrz&nska, M., Tomaszewksi, M., Grzelczak, Z., Rejman, E., Buchowicz, J.: Cascade activation of genome transcription in wheat. Nature (Lond.) New Biol. 244, 507-509 (1973) Grierson, D., Smith, H.: The synthesis and stability of ribosomal RNA in blue-green algae. Europ. J. Bioehem. 86, 280-285 (1973) Guinn, G.: Extraction of nucleic acids from lyophilized plant material. Plant Physiol. 41, 689-695 (1966) Jakob, ](. M., Bovey, F.: Early nucleic acid and protein syntheses in the primary root tips of germinating Vicia/aba. Exp. Cell Res. 54, 118-126 (1969) Julien, R., Grellet, F., Guitton, Y. : Synth~se des RNA au cours de la germination du radis. Physiol. Plantarum (Kbh.) 28, 323-324 (1970) Loening, U. E. : The fractionation of high-molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102, 251-257 (1967) Loening, U. E., Ingle, J. : Diversity of RNA components in green plant tissues. Nature (Lond.) 215, 363-367 (1967) Melera, P. W. : Nucleic acid metabolism in germinating onion. I. Changes in root tip nucleic acid during germination. Plant Physiol. 48, 73-81 (1971) Nygaard, P.: Deoxyribonucleotide pools in plant tissue cultures. Physiol. Plantarum (Kbh.) 26, 29-33 (1972) Trewavas, A. : The turnover of nculeic acids in Lemna minor. Plant Physiol. 45, 742-751 (1970) Van de Walle, C. : MAK column chromatography of the first RNA synthesized during germination of Zea mays embryos. ~EBS Lett. 16, 219-221 (1971) Walbot, V.: Rate of RNA synthesis and tRNA end-labelling during early development of Phaseolus. Planta (Berl.) 108, 161-171 (1972)

Onset of nucleic acid synthesis during germination of Pisum sativum L.

Measurments of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximatel...
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