PHYSIOLOGIA PLANTARUM 115: 155–165. 2002 Copyright C Physiologia Plantarum 2002

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Pyrimidine nucleotide and nucleic acid synthesis in embryos andmegagametophytes of white spruce (Picea glauca) during germination

Claudio Stasollaa,1, Natalia Loukaninaa, Hiroshi Ashiharab, Edward C. Yeunga and Trevor A. Thorpea,*

aPlant Physiology Research Group, Department of Biological Sciences, University of, Calgary, Calgary, Alberta T2N 1N4, CanadabDepartment of Biology, Faculty of Science, Ochanomizu University, Tokyo 112–8610, Japan1Present address: NC State, Forest Biotechnology Group, 2500 Partners II Bldg., Raleigh, NC 27695, USA*Corresponding author, e-mail: tthorpe/ucalgary.ca

Received 23 May 2001; revised 8 October 2001; in final form 26 October 2001

Pyrimidine nucleotide synthesis was investigated in isolatedgerminating zygotic embryos and separated megagametophyt-es of white spruce by following the metabolic fate of 14C-labelled orotic acid, uridine, and uracil, as well as by measur-ing the activities of the major enzymes participating in nucle-otide synthesis. The rate of nucleic acid synthesis in thesetissues was also examined by tracer experiments and autora-diographic studies conducted with labelled thymidine, and byconventional light microscopy. From our results, it emergesthat changes in the contribution of the de novo and salvagepathways of pyrimidines play an important role during theinitial stages of zygotic embryo germination. Preferential util-ization of uridine for nucleic acid synthesis, via the salvagepathway, was observed at the onset of germination, before therestoration of a fully functional de novo pathway. Similar

Introduction

White spruce is an important coniferous species inNorth America, especially for its use in pulp and lum-ber production (Farrar 1996). In recent years, the es-tablishment of an efficient regeneration system via so-matic embryogenesis (Lu and Thorpe 1987) has consti-tuted a valuable tool for the propagation of thisspecies, as well as a good system which has allowedcomparative studies of in vivo and in vitro embryogen-esis. Today, in fact, our studies have increased knowl-edge on the structural, physiological, and biochemicalevents occurring during zygotic and somatic embryo-genesis (Joy et al. 1991, 1997, Kong and Yeung 1992,1995, Kong et al. 1997, 1999, Yeung et al. 1998, Sta-solla and Yeung 1999, 2001, Ashihara et al. 2000,2001a,2001b, Stasolla et al. 2001a,2001b, 2001c). Be-

Abbreviations – DTT, dithiothreitol; OPRT, orotate phosphoribosyltransferase; PRPP, 5-phosphoribosyl-1-pyrophosphate; NPT, nucleoside phospho-transferases; TK, thimidine kinase, UPRT, uracil phosphoribosyltransferase; UK, uridine kinase.

Physiol. Plant. 115, 2002 155

metabolic changes, not observed in the gametophytic tissue,were also documented in somatic embryos previously. Thesealterations of the overall pyrimidine metabolism may repre-sent a strategy for ensuring the germinating embryos with alarge nucleotide pool. Utilization of 14C-thymidine for nucleicacid synthesis increased in both dissected embryos and mega-gametophytes during germination. Autoradiographic and lightmicroscopic studies indicated that soon after imbibition, DNAsynthesis was preferentially initiated along the embryonicaxis, especially in the cortical cells. Apical meristem reacti-vation was a later event, and the root meristem became acti-vated before the shoot meristem. Taken together, these resultsindicate that precise changes in nucleotide and nucleic acidmetabolism occur during the early phases of embryo germi-nation.

sides its theoretical significance, knowledge on zygoticembryo maturation and germination represents a valu-able tool for improving the somatic embryogenic pro-cess via the design of rational media and improvedculture conditions. This is particularly true for post-embryonic growth, as failure to germinate and convert(emergence of root and new leaf primordia) is oftenobserved in somatic embryos. Such a failure has beenascribed to the lack of resumption of mitotic activityin the apical meristems (Kong and Yeung 1992). Thus,comparative studies on nucleic acid metabolism andcell reactivation in germinating zygotic and somaticembryos might provide insights into the causes of thelow conversion frequency observed in the latter.

Pyrimidine nucleotides have important functions,

Fig. 1 The probable metabolic fate of 14C-labelled orotic acid, uridine, uracil, andthymidine in embryos andmegagametophytes of white spruce duringgermination. Enzymes measured areenclosed in boxes. OPRT, orotic acidphosphoribosyltransferase; UPRT, uracilphosphoribosyltransferase; NPT,nucleoside phosphotransferase; UK,uridine kinase; TK, thymidine kinase.

which are closely related to the growth and developmentof plants. Besides being building blocks for nucleic acidbiosynthesis, pyrimidine nucleotides participate in bio-energetic processes, as well as in the synthesis of macro-molecules, such as sucrose, polysaccharides, phospho-and glyco-lipids and several secondary products (see Ross1981). Catabolism of pyrimidines has also been associ-ated with the synthesis of nitrogen compounds. Severalstudies conducted in plant systems have revealed the pres-ence of two independent pathways for the synthesis ofpyrimidine nucleotides (King et al. 1965, Ross and Cole1968, Ashihara 1977). In the de novo pathway, UMP isgenerated from amino acids and other small molecules,whereas in the salvage pathway preformed bases andnucleosides are utilized as precursors (see Ross 1981).UMP produced by these pathways is then utilized for nuc-leic acid synthesis. Degradation of pyrimidines occurs viaindependent pathways (Fig. 1). Relative changes of thesepathways often relate to differentiation and development(Nygaard 1973, Ashihara et al. 2000). In our previous

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studies we demonstrated that these pathways are operat-ive in white spruce cultured cells (Ashihara et al. 2000),and that changes in their contribution to the overall nucle-otide pool occur during the maturation and germinationof white spruce somatic embryos (Ashihara et al. 2001b,Stasolla et al. 2001a,2001c). During somatic embryo ger-mination a tight regulation of the de novo and salvagepathway was required for the enlargement of nucleotidepool. Specifically, the operative uridine salvage observedduring the initial phases of germination was required forproviding the somatic embryos with sufficient pyrimidinenucleotides, before the restoration of the de novo pathway(Stasolla et al. 2001c).

As a part of our long-term comparative studies be-tween somatic and zygotic embryogenesis in whitespruce, it was our objective to determine whether similarchanges in pyrimidine metabolism also occur in germin-ating zygotic embryos. Dissected embryos and megaga-metophytes from germinating seeds were analysed separ-ately. This was necessary to ascertain whether the mega-

gametophytic tissue affects/regulates the metabolism ofpyrimidine nucleotides of the embryos, as reported forother metabolic processes (Stone and Gifford 1999).Based on previous results (Ashihara et al. 2000), pyrim-idine metabolism was investigated by following themetabolic fate of 14C-labelled orotic acid, uridine, anduracil as markers for the de novo, salvage, and degrada-tion pathways, respectively (see Fig. 1). Nucleic acid syn-thesis was monitored by following the metabolic fate of14C-labelled thymidine and by autoradiographic studiesusing 3H-labelled thymidine. In addition, the activitiesof the major key enzymes involved in the synthesis ofpyrimidine nucleotides were also measured.

Materials and methods

Radiochemicals

[2–14C]uracil (specific activity, 1.85 MBq mmolª1), [2–14C]uridine (2.00 MBq mmolª1), [2–14C]orotic acid (1.85MBq mmolª1), [2–14C]thymidine (specific activity, 1.85MBq mmolª1), and [methyl-3H]thymidine (specific activ-ity, 2.2 MBq mmolª1) were obtained from Moravek Bio-chemicals Inc. (Brea, CA, USA).

Plant materials

White spruce (Picea glauca [Moench] Vos) seeds (lot .7431580.1; germinability 92%) were obtained from theNational Tree Seed Centre, Fredericton, NB, Canada.Seeds were soaked in water at 4æC for 24 h, sterilized in25% Javex bleach for 20 min, and germinated onmoistened filter papers in the dark at 22æC. Germinatedseeds were collected at day 3, 6, and 12 and the embryoswere dissected out. Both isolated embryos and megaga-metophytes enclosed in their seed coats were processedfor tracing experiments and enzyme assays. For day 0,embryos were dissected from dry seeds. As the seed coatwas mainly composed of dead tissue, and represented asmall fraction of the seed, it was handled as part of themegagametophyte.

Metabolism of labelled pyrimidine precursors

Administration of labelled compounds into germinatingzygotic embryos and megagametophytes on the day indi-cated was carried out according to the procedure de-scribed in a previous paper (Ashihara et al. 2000). Tissue(approximately 50 dissected embryos and megagameto-phytes) was incubated in the presence of 10 ml of labelledcompounds for 2 h at 22æC. Extraction and analysis oflabelled metabolites was performed as described byAshihara et al. (2000). The samples were extracted suc-cessively with cold 6% perchloric acid (PCA) (PCA-sol-uble fraction), a mixture of ethanol and ether (1 : 1, v/v)at 50æC for 15 min (lipid fraction), and 6% PCA at100æC for 20 min (DNA plus RNA fraction). The sum-mation of the radioactivity found in these different frac-tions was designated total uptake.

Physiol. Plant. 115, 2002 157

Preparation and assay of enzymes

For preparation of enzymes, 400 embryos and megaga-metophytes were utilized. Extraction and determinationof specific activities of orotate phosphoribosyltransfera-se (OPRT), uracil phosphoribosyltransferase (UPRT),uridine kinase (UK), and nucleoside phosphotransferasemeasured with uridine (NPT uridine), were performedas described by Ashihara et al. (2000). Activities of thy-midine kinase (TK) and nucleoside phosphotransferasemeasured with thymidine (NPT thymidine) were carriedout as reported by Mullin and Fites (1978).

Light microscopy and autoradiography

For light microscopic studies, germinating embryos andmegagametophytes were fixed in 2.5% glutaraldehydeand 1.6% paraformaldehyde buffered with 0.05 M phos-phate buffer, pH 6.9, dehydrated with methyl cellosolve,followed by two changes of absolute ethanol, and theninfiltrated and embedded in Technovit 7100 (Kulzer andCo. Gmbh, Bereich Technik, D-6393 Wehrheim, Ger-many) (Yeung 1999). Sectioning was carried out withglass knives on a Reichert-Jung 2040 Autocut micro-tome. Serial longitudinal sections were cut at a thicknessof 3 mm. For general histological examinations, the sec-tions were stained with the periodic acid-Schiff (PAS)reaction and counterstained with 0.05% (w/v) toluidineblue O in benzoate buffer, pH 4.4 (Yeung 1984a). Thepreparations were examined and photographed with aLeitz Aristoplan light microscope.

Autoradiography of [methyl-3H]thymidine was carriedout as described by Yeung (1984b). Germinating em-bryos and megagametophytes were incubated in thepresence of [methyl-3H]thymidine (1.5 mCi mlª1) for 18h, fixed in FAA (Formaldehyde-ethanol-acetic acid[10%-50%-5%]), dehydrated using TBA (tertiary buta-nol) series, and embedded in Paraffin (Oxford Labware,St. Louis, MO, USA). Serial longitudinal sections werecut at a thickness of 7 mm. The selected slides were de-waxed and dipped into autoradiographic emulsion (Ko-dak NTB2, Canada Inc., Toronto, ON, Canada) dilutedto half the original concentration in distilled water.After a 10-d exposure at 4æC, development and fixationwere performed with Kodak D-19 developer (Kodak D-19, Canada Inc., Toronto, ON, Canada) and Kodak fixer(Kodak, Canada Inc., Toronto, ON, Canada) accordingto the procedure described by Yeung (1984b) The tissuewas stained in a 0.1% TBO (toluidine blue O solution),examined and photographed with a Leitz Aristoplanlight microscope.

Results

Changes of pyrimidine metabolism in isolated germinat-ing white spruce zygotic embryos and their separatedmegagametophytes were investigated by following themetabolic fate of several pyrimidine precursors and bymeasuring the activities of major key enzymes.

Fig. 2 Total uptake of pyrimidineprecursors in isolated embryos andmegagametophytes of white spruce atdifferent stages of germination. Values,expressed as pmol embryoª1 ormegagametophyteª1, are means ∫SE. OA,orotic acid; U, uracil; UR, uridine; TdR,thymidine.

Uptake of labelled precursors

Total uptake of the supplied 14C-labelled pyrimidine pre-cursors into the zygotic embryos and megagametophytesduring germination was estimated by adding the radio-activity recovered in the PCA, nucleic acid, ethanol-ether, and CO2 fractions. The uptake for all precursorswas low in dry seeds, and it gradually increased as ger-mination progressed (Fig. 2). Absorption of 14C-uracilwas generally the highest, whereas that of 14C-oroticacid was the lowest. When compared to that of the me-gagametophytes, the uptake values of all pyrimidine pre-cursors in the embryos were lower, especially during theinitial days of germination. As germination progressed,higher uptake values for orotic acid and uracil were ob-served in the embryos (Fig. 2).

Pyrimidine metabolism

The pyrimidine de novo pathway of the embryos andmegagametophytes during germination was investigatedby following the metabolic fate of 14C-labelled oroticacid (see Fig. 1). As shown on Table 1, after 2 h incuba-tion a large proportion (51.2%) of orotic acid taken upby the dried embryos was recovered as nucleotides(UMP π UDP π UTP). Only small traces of radioac-tivity (1.6%) were recovered as nucleic acids. As germi-nation progressed, the contribution of orotic acid toboth nucleotide and nucleic acid synthesis increased.

Physiol. Plant. 115, 2002158

After 12 days in germination, almost 94% of radioac-tivity taken up by the embryos was incorporated intonucleotides (70.9%) and nucleic acids (22.3%). Com-pared to the embryos, the percentage of label from [2–14C]orotic acid recovered into the nucleic acid fractionof megagametophytic tissue was higher (8.8%) at day 0,and did not change during the following days of germi-nation. An increasing proportion of radioactivity recov-ered in the nucleotide fraction was observed as germi-nation progressed (9.1% at day 0 and 23.3% at day 12).

The activity of the pyrimidine salvage pathway wasassayed by following the incorporation of [2–14C]uridineinto salvage products, namely nucleotides and nucleicacids (see Fig. 1). After 2 h incubation, 66.1% of labelleduridine was salvaged in dried embryos (58% as nucleo-tides and 8.1% as nucleic acids). This percentage in-creased during germination. At day 12, 66.9% of theradioactivity was recovered as nucleotides, and 21.2% asnucleic acids. Lower salvage activity was observed in themegagametophytes, where the percentage of uridine sal-vaged was 14.7% at day 0, and 45.3% at day 12. Withinthis percentage, a larger proportion of the label was re-covered as nucleotides, especially at day 12 (Table 1).

The degradation pathway of pyrimidine metabolismwas investigated by following the metabolic fate of [2–14C]uracil (see Fig. 1). As shown in Table 1, more than57% of the fed precursor was degraded in the embryosthroughout the 12 days of germination. At day 0 themost labelled metabolite was b-ureidopropionate (al-most 50%), whereas at day 12 a larger proportion of

Table 1. Metabolism of pyrimidine precursors during the initialstages of germination in isolated zygotic embryos and separatedmegagametophytes of white spruce. The rate of incorporation intothe major labelled fractions ∫SE is expressed as a percentage ofradioactivity taken up by the tissue. The embryos and megagameto-phytes were incubated for 2 h in the presence of radiolabelled pre-cursors

Day 0 Day 3 Day 6 Day 12

EMBRYODe novo synthesis[6–14C]orotic acidNucleotides 51.2 ∫ 5.5 50.7 ∫ 4.0 67.1 ∫ 4.2 70.9 ∫ 2.0Nucleic acids 1.6 ∫ 0.2 27.4 ∫ 1.4 16.6 ∫ 1.9 22.3 ∫ 1.6Total 52.8 ∫ 5.6 78.1 ∫ 5.2 83.7 ∫ 6.1 93.2 ∫ 3.6

Salvage synthesis[2–14C]UridineNucleotides 58.0 ∫ 2.6 64.7 ∫ 3.0 66.9 ∫ 5.5 66.9 ∫ 4.0Nucleic acids 8.1 ∫ 0.7 27.9 ∫ 1.9 23.8 ∫ 0.6 21.2 ∫ 1.3Total 66.1 ∫ 3.4 92.6 ∫ 4.9 90.7 ∫ 6.1 88.1 ∫ 2.7

Degradation[2–14C]Uracilb-Ureidopropionate 49.8 ∫ 3.2 7.2 ∫ 1.0 3.2 ∫ 0.8 6.6 ∫ 0.4CO2 17.5 ∫ 1.0 49.8 ∫ 2.1 55.5 ∫ 3.2 51.1 ∫ 2.3Total 67.2 ∫ 4.3 57.0 ∫ 3.1 58.7 ∫ 4.0 57.7 ∫ 2.7

MEGAGAMETOPHYTEDe novo synthesis[6–14C]orotic acidNucleotides 9.1 ∫ 0.7 18.9 ∫ 2.0 20.6 ∫ 1.4 23.3 ∫ 3.2Nucleic acids 8.8 ∫ 1.9 10.8 ∫ 1.1 14.9 ∫ 0.4 9.7 ∫ 1.1Total 17.9 ∫ 2.7 29.7 ∫ 3.1 35.5 ∫ 1.8 33.0 ∫ 4.3

Salvage synthesis[2–14C]Uridine 9.3 ∫ 0.3 18.4 ∫ 1.9 14.5 ∫ 0.5 37.2 ∫ 3.1NucleotidesNucleic acids 5.4 ∫ 1.5 11.9 ∫ 2.4 11.4 ∫ 1.3 8.1 ∫ 0.4Total 14.7 ∫ 1.2 30.3 ∫ 4.3 25.9 ∫ 0.7 45.3 ∫ 3.5

Degradation[2–14C]Uracilb-Ureidopropionate 9.4 ∫ 0.8 2.7 ∫ 0.3 1.9 ∫ 0.1 3.0 ∫ 0.4CO2 17.2 ∫ 2.2 25.3 ∫ 1.6 28.6 ∫ 0.2 29.1 ∫ 0.3Total 26.6 ∫ 1.3 28.0 ∫ 1.2 30.5 ∫ 0.1 32.1 ∫ 0.7

radioactivity was released as CO2 (almost 52%). In themegagametophyte, the percentage of [2–14C]uracil de-graded was lower than 33% during the course of theexperiment. CO2 was the most labelled degradationproduct recovered from megagametophytic tissue.

Nucleic acid synthesis during germination was investi-gated by following the metabolism of [2–14C]thymidine(see Fig. 1). As shown in Table 2, an increasing amountof thymidine taken up by the embryos was catabolizedas b-ureidoisobutyric acid and CO2 during the first 12days of germination (29.3% at day 0 and 78.9% at day12). This increase paralleled a decrease of radioactivityrecovered in unmetabolized precursor (51.8% at day 0and 2.3% at day 12). Anabolism of [2–14C]thymidine waslow at all stages of embryo germination. Incorporationof radioactivity into nucleotides was high in dried em-bryos and it declined as germination progressed. In con-trast, utilization of thymidine for nucleic acid synthesisincreased (Table 2). A lower percentage of fed thymidinewas catabolized in the megagametophyte (11.7% at day

Physiol. Plant. 115, 2002 159

0 and 24.9% at day 12). More than 57% of radioactivitywas recovered as unmetabolized precursor throughoutthe course of the experiment. Less than 10% of fed thy-midine was anabolized by megagametophytic tissuethroughout the course of the experiment (Table 2).

Activities of the enzymes involved in pyrimidinemetabolism

The specific activity of orotate phosphoribosyltransfera-se (OPRT), a key enzyme of the de novo pyrimidine bio-synthetic pathway (see Fig. 1), was high in the embryosand low in the megagametophytes (Fig. 3). The activityof uridine kinase (UK), the enzyme responsible for thesalvage of uridine, was high in dried embryos and itsharply decreased during the first days of germination.Fluctuation in the activity of this enzyme was observedin the megagametophyte. The activity of uracil phos-phoribosyltranferase (UPRT), which converts uracil toUMP, was lower than that measured for OPRT and UKfor both embryos and megagametophytes at all stagesof germination. In the embryos, a peak in the activity ofthis enzyme was observed at day 3. Low levels of activityof nucleoside phosphotransferase measured for uridine(NPT uridine) were observed in both embryos and me-gagametophytes. No activity of thymidine kinase (TK),the enzyme which catalyses the conversion of thymidineto TMP, was detected at days 0 and 3 in either embryosor megagametophytes. The activity of this enzyme in-creased during the following stages of germination.Nucleoside phosphotransferase activity measured forthymidine (NPT thymidine) was also detected duringgermination. Higher levels of this enzyme were measuredfor the embryos compared to the megagametophytictissue, especially at day 0 and day 12 (Fig. 3).

Light microscopic and autoradiographic study

Dry embryos of white spruce were characterized by welldeveloped shoot and root apical meristems, located atthe apical poles of the embryonic axis (Figs 4A,B).Initiation of cell division was observed around day 3along the embryonic axis, especially in cortical cells, butnot in the procambial cells which continued to elongate(Fig. 4C). Mitotic activity in the apical meristems wasonly a later event, as it was observed around day 6 inthe cells of the root apical meristem (Fig. 4D), andaround day 8 in those of the shoot meristem (Fig. 4E).After day 10, cell division was mainly observed alongthe embryonic axis (Fig. 4F). No mitotic activity was de-tected in the megagametophyte at any stage of germi-nation (data not shown).

Thymidine incorporation in the nuclei of the embryoswas found to start soon after imbibition. At day 3, al-though no incorporation was observed in the cells of theapical meristems, many cortical cells in proximity of theroot pole, were heavily labelled with [methyl-3H]thymidi-

Table 2 Thymidine metabolism during the initial stages of germination in isolated zygotic embryos and separated megagametophytes ofwhite spruce. The rate of incorporation into the major labelled fractions ∫SE is expressed as a percentage of radioactivity taken up by thetissue. The embryos and megagametophytes were incubated for 2 h in the presence of radiolabelled thymidine

Day 0 Day 3 Day 6 Day 12

EMBRYOThymidine anabolismdTTP π dTDT π dTMP 6.8 ∫ 0.4 4.3 ∫ 0.2 2.2 ∫ 0.1 1.5 ∫ 0.0Nucleic acids 1.1 ∫ 0.0 3.4 ∫ 0.5 5.3 ∫ 0.1 7.8 ∫ 0.1Total 7.9 ∫ 0.3 7.7 ∫ 0.5 7.5 ∫ 0.2 9.3 ∫ 0.1

Thymidine catabolismb-Ureidoisobutyric acid 2.0 ∫ 0.1 1.1 ∫ 0.0 0.8 ∫ 0.1 2.3 ∫ 0.3CO2 27.3 ∫ 3.0 23.8 ∫ 2.1 82.0 ∫ 1.2 76.6 ∫ 1.9Total 29.3 ∫ 3.2 24.9 ∫ 2.1 82.8 ∫ 1.3 78.9 ∫ 1.6

Unmetabolized thymidine 51.8 ∫ 3.0 58.1 ∫ 3.9 5.9 ∫ 0.3 2.3 ∫ 0.3MEGAGAMETOPHYTEThymidine anabolismdTTP π dTDT π dTMP 1.3 ∫ 0.1 1.4 ∫ 0.1 1.5 ∫ 0.2 2.4 ∫ 0.3Nucleic acid 2.3 ∫ 0.1 3.1 ∫ 0.1 8.2 ∫ 0.3 5.2 ∫ 0.4Total 3.6 ∫ 0.2 4.5 ∫ 0.1 9.7 ∫ 0.4 7.6 ∫ 0.6

Thymidine catabolismb-Ureidoisobutyric acid 0.4 ∫ 0.0 0.2 ∫ 0.0 0.1 ∫ 0.0 0.4 ∫ 0.1CO2 11.3 ∫ 0.2 15.6 ∫ 2.0 18.6 ∫ 1.6 24.5 ∫ 0.6Total 11.7 ∫ 0.2 15.8 ∫ 2.0 18.7 ∫ 1.6 24.9 ∫ 0.5

81.6 ∫ 6.5 78.2 ∫ 3.2 59.9 ∫ 3.1 57.2 ∫ 4.2Unmetabolized thymidine

Fig. 3. Specific activities of the enzymesparticipating in the synthesis ofpyrimidine nucleotides in isolatedembryos and megagametophytes of whitespruce during germination. Values,expressed as pkat mgª1 protein, aremeans ∫ . OPRT, orotic acidphosphoribosyltransferase; UPRT, uracilphosphoribosyltransferase; UK, uridinekinase; TK, thymidine kinase; NPT,nucleoside phosphotransferase measuredwith uridine or thymidine.

Physiol. Plant. 115, 2002160

Fig. 4. Micrographs showing the cell division pattern in germinating embryos of white spruce. Dried embryos were characterized by a dome-shaped shoot meristem composed by an apical layer of large cells (arrowhead) and a subapical region (*), where storage products accumulated(A). Large isodiametrical cells which stained less than the surrounding cells formed the root meristem (arrowheads) (B). Cell divisions(arrowheads) were first observed at day 3 in the cortex cells of the embryonic axis. No mitotic activity was observed in the procambial cells(*) which started to elongate (C). Mitotic figures (arrowheads) were first observed in the root apical meristem at day 6 (D) and in the shootapical meristems around day 8 (E). During the subsequent days of germination, cell divisions (arrowheads) were localized along the embry-onic axis (F). Scale bars Ω 40 mm.

ne (Figs 5A,B). At day 6, incorporation of thymidine oc-curred mainly in the cells of cotyledons and cortex. Noincorporation was observed in the shoot apical meristem(Fig. 5C). Many cells in the root pole were labelled atday 6 (Fig. 5D). Reactivation of the shoot apical meris-

Physiol. Plant. 115, 2002 161

tem, as revealed by the accumulation of silver grains,was only observed at day 12 (Fig. 5E). At this stage fewerroot cells were labelled (Fig. 5F). No label was observedin megagametophytic cells throughout the course of theexperiment.

Fig. 5. Autoradiographs showing the incorporation of [methyl-3H]thymidine in shoot and root poles of germinating white spruce zygoticembryos. After 3 days of germination, no thymidine was incorporated in the cells of the shoot apical meristem (arrowhead) (A). Incorpor-ation of [methyl-3H]thymidine was mainly observed in the nuclei of cortical cells of the roots. No label was however, observed in the cellsof the root apical meristem (arrowheads) (B). At day 6, although not detected in the shoot apical meristem (arrowhead), thymidine incorpor-ation was observed in the cotyledons (arrows) and along the embryonic axis (C). At this stage, an intense accumulation of silver grains wasalso noticed within the cells of the root apical meristem (arrowheads) (D). After 12 days, cells of the shoot meristem (arrowhead) becamemitotically active, as indicated by the labelled nuclei (E). At this stage, only a few cells in the root pole were found to incorporate thymidine(F). Scale bars Ω 200 mm.

Discussion

Pyrimidine nucleotide and nucleic acid synthesis were in-vestigated in germinating white spruce zygotic embryosand megagametophytes, by following the metabolic fateof 14C-labelled precursors and by autoradiographic

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studies. As shown in Fig. 2, the uptake of all pyrimidineprecursors is low in both dried embryos and megagame-tophytes, but increases sharply as germination pro-gresses. The reduced ability of dried seeds to metabolizepyrimidine bases and nucleosides is indicative of a reduc-tion of the overall cellular metabolism. Similar low up-

take values were also reported in partially dried whitespruce somatic embryos fed with 14C-labelled purinesand pyrimidine precursors (Stasolla et al. 2001b,2001c),and in dry Sinapia arvensis seeds incubated with 14CO2

(Edwards 1976).Incorporation of radioactivity from orotic acid into

nucleotides and nucleic acids clearly indicates that thepyrimidine de novo pathway is active in dried seeds, espe-cially in the embryos, and increases as germination pro-gresses. Partially dried white spruce somatic embryoswere also endowed with the de novo nucleotide bio-synthetic machinery (Stasolla et al. 2001a), although dif-ferent results were reported by Ashihara (1977), with ger-minating black gram embryos. It is worth mentioningthat at day 0, orotic acid utilization for nucleic acid syn-thesis in the megagametophytes is four times higher thanthat observed in the embryos. This higher percentage ofincorporation suggests a faster turnover of de novo pro-duced nucleotides, which are utilized as building blocksfor nucleic acid synthesis. Active transcription in gameto-phytic tissue during the initial stages of germination is animportant event, which is required for the mobilization ofstorage products in support of embryo growth. The roleplayed by the megagametophyte as a nourishing tissueduring conifer seed germination is well documented in theliterature (Stone and Gifford 1999).

As germination progresses, the activity of the de novopathway increases in the embryos (Table 1). Increased or-otate anabolism, also reported in white spruce somaticembryos (Stasolla et al. 2001c), black gram seeds (Ashi-hara 1977) and Alaska peas cotyledons (Ross and Mur-ray 1971, Ross et al. 1971), may be required for the en-largement of nucleotide pool necessary to support thegrowth of the germinating embryo.

Contribution of uridine to nucleotide and nucleic acidbiosynthesis through the salvage pathway (Fig. 1) wasalso operative in dried white spruce seeds (Table 1).Compared to orotic acid, however, uridine seems to bethe preferential precursor for nucleic acids at the incep-tion of germination. The predominant role of the sal-vage pathway, compared to the de novo pathway, duringthe initial stages of germination was also reported in so-matic embryos of white spruce (Stasolla et al. 2001c),and in zygotic black gram embryos (Ashihara 1977).The differential contribution of the two pathways to nu-cleotide and nucleic acid synthesis may represent a com-mon strategy for seeds to produce a sufficient nucleotidepool during the initial stages of germination. Active sal-vage at the inception of germination might in fact benecessary before the de novo machinery becomes fullyoperative. This hypothesis is also confirmed by the activ-ity of uridine kinase (UK), the enzyme responsible forthe salvage of uridine, which is high in dried embryosand it declines as germination progresses (Fig. 3). Highactivity of UK was also observed during the lag phaseof Vinca rosea cultured cells, characterized by a highsalvage activity, in preparation for the cell division phase(Kanamori-Fukuda et al. 1981). Compared to zygoticembryos, the salvage pathway in the megagametophytes

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was much lower throughout the course of the experi-ment. Since UK activity in the megagametophytic tissueis always higher than that measured in the embryos afterthe first days of germination (Fig. 3), factors other thanUK availability might explain the differential utilizationof uridine in the megagametophyte.

An operative degradation pathway, as measured by theincorporation of 14C-labelled uracil into b-ureidopro-pionate and CO2 (Fig. 1) was also observed in both em-bryos and megagametophytes at all stages of germi-nation (Table 1). Active catabolism of uracil, previouslydocumented during white spruce somatic embryo matu-ration (Ashihara et al. 2001b) and germination (Stasollaet al. 2001c), was also reported in other systems, includ-ing Catharanthus roseus cells (Kanamori-Fukuda et al.1981) and in germinating black gram seeds (Ashihara1977). The extensive anabolism of uracil in white spruceis mainly ascribed to the low activity of uracil phosphor-ibosyltransferase (UPRT), compared to UK, the salvageenzyme for uridine (Fig. 3).

The contribution of both salvage and the novo path-ways to nucleotide biosynthesis is required to sustainDNA replication and cell division at the inception ofgermination. Such events were investigated by utilizinglabelled thymidine in tracing experiments and autora-diographic studies. In both embryos and megagameto-phytes, a large fraction of exogenously supplied thymi-dine was either-non-metabolized, especially during theinitial phases of germination, or catabolized (Table 2).Poor utilization of thymidine for deoxyribonucleotideand DNA synthesis was also observed during whitespruce somatic embryogenesis (Stasolla et al. manu-script in preparation) and in germinating black gramseeds (Kameyama et al. 1985). Turnover of thymidine isslow in dry white spruce embryos, as a large proportionof radioactivity is recovered as deoxyribonucleotides,but increases upon imbibition. During germination, infact the depletion of the deoxyribonucleotide fractionparallels an increase in radioactivity recovered as nucleicacids (Table 2). Autoradiographic and light microscopicstudies also suggest that active DNA synthesis and celldivision occur during the early stages of germination,especially along the embryonic axis, but not in the meris-tematic regions (Figs 4C and 5A,B). Initiation of cell di-vision in the embryonic axis, together with cell expan-sion, is most likely required for radicle emergence, whichin white spruce seeds occurs between days 5 and 9. Asalso reported by Yeung et al. (1998), the reactivation ofthe root apical meristem of white spruce zygotic em-bryos occurs around day 6 (Figs 4D and 5D), whereasthat of the shoot apical meristem is only observed later(Figs 4E and 5E). In contrast to the embryos, incorpor-ation of labelled thymidine into deoxyribonucleotidesand nucleic acids increases in the megagametophytictissue as germination progresses (Table 2).

The last point emerging from this study concerns theactivity of the enzymes involved in thymidine anabolism.In dried white spruce seeds and during the first 3 daysof germination nucleotide phosphotransferase (NPT

thymidine) is the only enzyme involved in the conversionof thymidine to TMP. Thymidine kinase (TK) activity,in fact, is only detectable at later stages of germination(Fig. 3). Absence of TK activity during the initial phasesof germination was also reported in other systems(Schwarz and Fites 1970, Kameyama et al. 1985). Theactivity of this enzyme seems to be strictly associatedwith the biochemical events culminating in DNA syn-thesis and cell division of the white spruce embryos. Inagreement with this idea, TK activity was only observedduring the S phase of the cell cycle of CEM culturedcells (Bianchi et al. 1997).

In conclusion, changes in the contribution of the denovo and salvage pathway of pyrimidine metabolismplay an important role during the initial stages of whitespruce zygotic embryo germination. An active utiliza-tion of uridine for nucleic acid synthesis through the sal-vage pathway is necessary at the onset of germination,before the activation of a fully functional de novo path-way. Such changes, also observed during somatic em-bryo germination, seem to be strictly associated with theinitiation of cell division and subsequent growth, as theyare not observed in the megagametophytic tissue. An op-erative pyrimidine metabolism is necessary to supportthe growth of the embryo, which occurs through a seriesof co-ordinated DNA synthesis and cell division events.Thus, the information obtained from this study indi-cates that the zygotic and somatic embryos undergosimilar changes in pyrimidine nucleotide and nucleicacid synthesis during the early stages of germination.

Acknowledgements – This research was supported by a NaturalSciences and Engineering Research Council of Canada (NSERC)scholarship to C. Stasolla and by NSERC Research Grants to E.C. Yeung, and T. A. Thorpe. H. Ashihara acknowledges with grati-tude the support from the latter grant, while a Visiting Scientist atthe University of Calgary in Fall 1998. The authors also thank theNational Tree Seed Centre (Fredericton, N.B., Canada), for provid-ing the white spruce seeds.

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