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Activation of the Cell Cycle in Tomato (Lycopersicon esculentum Mill.) Seeds during Osmoconditioning as Related to Temperature and Oxygen
Annals of Botany 84 : 245–251, 1999 Article No. anbo.1999.0916, available online at http:\\www.idealibrary.com on
Activation of the Cell Cycle in Tomato (Lycopersicon esculentum Mill.) Seeds during Osmoconditioning as Related to Temperature and Oxygen N. O$ Z B I N G O$ L*, F. C O R B I N E A U*†, S. P. C. G R O OT‡, R. J. B I N O‡ and D. C O# M E* * UniersiteT Pierre et Marie Curie, Laboratoire de Physiologie VeT geT tale AppliqueT e, Tour 53, 1er eT tage, 4 Place Jussieu, 75252 Paris CeT dex 05, France, ‡ CPRO-DLO, P.O. Box 16, 6700 AA Wageningen, The Netherlands Received : 8 March 1999
Returned for revision : 14 April 1999
Accepted : 4 May 1999
Using flow cytometric analyses of the nuclear DNA content, we studied the effects of various conditions of osmopriming on the activation of the cell cycle in embryo root tips of tomato (Lycopersicon esculentum ‘ Elko ’) seeds. In dry untreated seeds, 90n7 % of the nuclei revealed 2C signals. Priming of seeds in polyethylene glycol-8000 (PEG) improved the germination rate of seeds transferred onto water at 15 mC. This was associated with an increase in 4C signals when priming was carried out at k1n0 and k1n5 MPa. Priming at k2n0 MPa enhanced subsequent germination but had no effect on DNA replication. For temperatures during priming up to 25 mC, a positive linear correlation existed between the efficiency of the treatment, evaluated by the reciprocal of time to obtain 50 % germination at 15 mC, and the frequency of 4C nuclei or the 4C\2C values. Such a correlation did not exist when priming was performed at higher temperatures. At least 5 % oxygen in the atmosphere was required during priming for the induction of DNA replication and for the enhancement of subsequent germination. In the presence of 5i10−% and 10−$ NaN during priming, most of the cells were maintained with 2C DNA levels and the treatment had no $ stimulatory effect on germination. The results show a positive linear relationship between the frequency of 4C DNA nuclei or the 4C\2C ratio and the improving effect of priming. However, in suboptimal conditions of priming (k2n0 MPa or temperatures higher than 25 mC), the improvement of seed germination was not associated with the onset of DNA replication. # 1999 Annals of Botany Company Key words : Cell cycle, germination, osmopriming, oxygen, temperature, Lycopersicon esculentum, tomato.
INTRODUCTION Osmopriming consists of imbibing seeds in an osmotic solution at low water potential in order to control their water uptake and prevent radicle protrusion. Such treatment improves the germination of seeds of numerous species (Heydecker, Higgins and Turner, 1975 ; Guedes and Cantliffe, 1980 ; Brocklehurst and Dearman, 1983 ; Corbineau, Picard and Co# me, 1993 ; Bray, 1995). Primed seeds germinate in a wider range of temperatures (Bray, 1995) and are less sensitive to oxygen deprivation (Corbineau and Co# me, 1990 ; Corbineau et al., 1993 ; Smok et al., 1993 ; Corbineau, Picard and Co# me, 1994) than unprimed ones. The beneficial effect of priming has been associated with various biochemical, cellular and molecular events including synthesis of RNA and proteins (Bray et al., 1989 ; Dell’Aquila and Bewley, 1989 ; Davison and Bray, 1991 ; Bray, 1995). Priming is also known to enhance respiratory activity of seeds (Halpin-Ingham and Sundstrom, 1992 ; Chojnowski, Corbineau and Co# me, 1997) and, when applied to aged seeds, restores activities of enzymes involved in the cell detoxifying mechanisms such as superoxide dismutase, catalase and glutathione reductase (Bailly et al., 1997). Studies on the effects of osmopriming on DNA replication † For correspondence. Fax j33 1 44 27 59 27, e-mail corbi!ccr.jussieu.fr
0305-7364\99\080245j07 $30.00\0
have given inconsistent results. Only low DNA synthesis was found during priming in leek (Allium porrum L.) (Bray et al., 1989 ; Ashraf and Bray, 1993) and tomato (Lycopersicon esculentum Miller) (Coolbear and Grierson, 1979) seeds. In contrast, studies using flow cytometry have demonstrated that DNA replication was initiated in the radicle tip cells during osmopriming of seeds of tomato (Bino et al., 1992 ; Lanteri et al., 1994) and pepper (Capsicum annuum L.) (Lanteri et al., 1993, 1994 ; Saracco et al., 1995). An increase in 4C DNA content was also observed in sugarbeet (Beta ulgaris L.) seeds (Redfearn and Osborne, 1997). Induction of DNA replication has also been associated with an accumulation of β-tubulin (De Castro et al., 1995). In pepper and tomato seeds, a positive correlation was found between the induction of DNA replication, measured as the increase in 4C nuclei, and the efficiency of the osmotic treatment (Lanteri et al., 1994). The percentage of 4C nuclei started to increase after 3 d of priming in the radicle tips of tomato embryos (Bino et al., 1992) and after 10 d in pepper embryos (Lanteri et al., 1993). A reduction of the polyethylene glycol concentration used for the priming treatment resulted in a greater proportion of cells with 4C DNA content (Lanteri et al., 1994 ; Saracco et al., 1995). Nevertheless, Saracco et al. (1995) found that priming could improve germination of pepper seeds without an increase in 4C nuclei. The aims of the present work were : (1) to investigate # 1999 Annals of Botany Company
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
246
T 1. Effects of 3 or 7 d of priming at 25 mC with PEG solutions at k1n0, k1n5 or k2n0 MPa on the seed moisture content (dry weight basis) before redrying, the subsequent germination rate at 15 mC, expressed as the time to obtain 50 % germination (T ), the percentages of 2C and 4C nuclei of the radicle tips, and the 4C\2C ratio &! Conditions of priming Duration (d) 0 (control) 3
7
Water potential (MPa)
Moisture content (% d. wt)
T (h)
2C (%)
4C (%)
(4C\2C) i100
— k1n0 k1n5 k2n0 k1n0 k1n5 k2n0
9n4p0n4 77n3p4n2 70n2p4n3 64n8p8n2 76n0p2n4 68n8p1n7 67n6p3n8
130p10 50p1 52p2 70p5 19p1 40p2 48p4
90n7p0n4 77n2p1n0 86n9p0n6 93n8p0n3 63n6p3n8 89n0p0n2 93n0p0n9
7n0p2n0 22n6p0n8 13n0p0n5 6n2p0n5 28n8p1n3 10n9p0n5 6n8p0n2
7n7p1n8 29n3p1n3 15n0p0n2 6n6p0n6 45n3p4n8 12n2p0n8 7n3p0n3
&!
Control seeds were not primed. Data are means of three (flow cytometry and moisture content) or four measurements (T )ps.d.
&!
effects of conditions of priming (water potential, oxygen and temperature) on tomato seed germination and DNA replication ; and (2) to determine whether the beneficial effect of priming is correlated with an increase in 4C nuclei. MATERIALS AND METHODS Plant material Experiments were carried out with seeds of tomato (Lycopersicon esculentum Mill., ‘ Elko ’) supplied by Clause Semences (France). Seeds were stored dry in the open air at 20 mC and 55 % relative humidity before the experiments started. Osmopriming treatments Seeds were primed for 7 d on a layer of cotton wool imbibed with polyethylene glycol-8000 (PEG) solutions at k1n0, k1n5 and k2n0 MPa. The treatments were carried out at temperatures ranging from 10 to 35 mC (p0n5 mC). Concentrations of PEG solutions were calculated according to Michel and Kaufmann (1973). Priming of seeds in various oxygen tensions (mixtures of nitrogen and oxygen) was performed for 7 d at 25 mC—the optimal temperature for priming of tomato seeds (O$ zbingo$ l, Corbineau and Co# me, 1998)—according to the procedure developed by Co# me and Tissaoui (1968). Gas mixtures were obtained through capillary tubes by mixing compressed air and pure nitrogen. The atmospheres thus obtained were passed continuously through germination chambers at a constant flow rate (4 l h−"). They contained 0 (pure nitrogen), 1, 3, 5, 10 and 21 % oxygen (air). In order to study whether a particular level of respiratory activity was necessary to achieve priming, seeds were soaked for 7 d at 25 mC in polyethylene glycol solutions at k1n0 MPa containing various concentrations of the respiratory inhibitor NaN ranging from 10−& to 10−$ . $ After the osmotic treatments, seeds were rinsed for 30 s with distilled water and were dried for 3 d in the open air at
20 mC and 55 % relative humidity. After redrying, the moisture content of primed seeds was similar (8–9 % d. wt basis) to that of control untreated seeds. Dry weight was obtained by oven drying seeds at 105 mC for 3 d.
Germination assays Germination ability was tested in samples of 100 seeds placed in 9 cm diameter Petri dishes (25 seeds per dish, four replicates) on a layer of cotton wool moistened with distilled water. Germination assays were carried out at 15 mC in darkness. A seed was regarded as germinated when the radicle protruded through the seed coat. Germination counts were made daily up to 15 d. Results were expressed as the time to reach 50 % germination (T ) or the reciprocal &! of T (1\T ). Results presented are the means of the results &! &! obtained in four replicatesps.d.
Flow cytometry Amounts of nuclear DNA were quantified using radicle tips of embryos isolated from control unprimed seeds and from seeds primed for 7 d in various conditions as specified earlier. Radicle tips (1 mm) were dissected from seeds, dried for 2 d at 20 mC in the presence of silica gel and stored at k30 mC for later use. Embryo radicle tips were then chopped with a razor blade in 500 µl of nucleus-isolation buffer containing 0n2 mannitol, 10 m 2(-morpholino) ethanesulphonic acid, 10 m NaCl, 10 m KCl, 10 m spermine tetrahydrochloride, 2n5 m ethylenediamine-tetraacetic acid, 2n5 m dithiothreitol, 0n05 % v\v triton X-100 and 0n05 % NaN at pH 5n8 as previously described by Bino et al. $ (1993). The mixture and additional 500 µl nucleus-isolation buffer were sieved through 88 µm nylon mesh into the flowcytometer test tubes. Twenty µl of 1 mg ml−" propidium iodide solution (Molecular Probes, Eugene, OR, USA) were then added to allow measurement of amounts of nuclear DNA by fluorescence. For each sample, five radicle tips, representing about 5000 to 10 000 nuclei, were used and
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds flow-cytometric determinations were made at least in duplicate. The DNA content of isolated nuclei was measured about 10 min after sample staining, using a flow cytometer (Coulter Corp., Miami, FL, USA, Epics XL-MCL model). Excitation of DNA-bound propidium iodide was performed by a 488 nm Argon Ion Laser, and fluorescence was detected over the range 605–635 nm. MultiCycle for Windows Cell Cycle Analysis software version 3n0 (Phoenix, Flow System Inc., San Diego, CA, USA) was used for curve fitting of fluorescence frequency distributions. The DNA amount is
A 100
T50 (h)
80
60
40
20
0
5
10
15
20
25
30
35
40
247
proportional to the fluorescent signal and is expressed as an arbitrary C value in which the 1C value represents the DNA content of the unreplicated haploid chromosome complement. Results presented are the means of the percentages of nuclei with 2C DNA levels and those with 4C DNA levels.
RESULTS Effects of priming at arious water potentials Effects on seed germination of priming for 3 and 7 d at 25 mC with PEG solutions at k1n0, k1n5 and k2n0 MPa are presented in Table 1. The moisture content of the seeds increased with higher water potentials. No seeds germinated during the osmotic treatments (data not shown). In all cases, primed seeds germinated faster and the time to obtain 50 % germination (T ) was decreased compared to that of &! unprimed seeds. The improvement of germination was higher after 7 d of priming than after 3 d, and a water potential of k1n0 MPa was the most efficient. For this reason, in all the following experiments seeds were primed for 7 d with a PEG solution at k1n0 MPa. Table 1 also shows the effects of priming on nuclear DNA content of radicle tip cells. In dry unprimed seeds, 90n7 % of the nuclei gave 2C signals, indicating that the majority of cells were arrested at the G phase of the cell cycle. Priming " with PEG at k1n5 and k1n0 MPa resulted in an increase of 4C signals, which was more marked with the highest water potential. However, priming with PEG at k2n0 MPa had no effect on DNA replication. The 4C\2C ratio increased up to about 29 and 45 % after 3 and 7 d of priming at
B 50 50 25° 40 30
4C and 4C/2C (%)
4C and 4C/2C (%)
40
20
20° 25°
30 20° 15° 20
30°
10
15°
10° 35°
10 0
5
10 15 20 30 25 Priming temperature (°C)
35
35°
40
F. 1. Effects of temperature during priming for 7 d with a PEG solution at k1n0 MPa on the subsequent germination rate at 15 mC (expressed as the time to obtain 50 % germination : T ) (A) and on the &! percentage of 4C nuclei (#) and the 4C\2C ratio ($) (B). Means of three (flow cytometry) or four measurements (T ). Vertical bars denote &! the largest s.d. Correlation coefficients of the regression lines in B were between 0n90 and 0n98. The values of T , 4C and 4C\2C for control &! unprimed seeds were 130p10 h, 7n0p2n0 % and 7n7p1n8 %, respectively (Table 1).
30°
10°
0
0·01
0·02
0·03 0·04 1/T50 (h–1)
0·05
0·06
F. 2. Relationships between the percentages of 4C nuclei (#) or the values of the 4C\2C ratio ($) and the subsequent germination rate at 15 mC, expressed as the reciprocal of time to obtain 50 % germination (T ). Seeds were primed for 7 d at 10, 15, 20, 25, 30 and 35 mC with a &! PEG solution at k1n0 MPa. Values correspond to the mean values given in Fig. 1.
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
248 A
50
150
21% 125 4C and 4C/2C (%)
40
T50 (h)
100
75
30
10%
20 10%
50 10
25
0
5
10
15
20
B
0
3% 0%
5% 1% 0·01
0·02
0·03 0·04 1/T50 (h–1)
0·05
0·06
F. 4. Relationships between the percentages of 4C nuclei (#) or the values of the 4C\2C ratio ($) and the subsequent germination rate at 15 mC expressed as the reciprocal of time to obtain 50 % germination (T ). Seeds were primed for 7 d at 25 mC with a PEG solution at &! k1n0 MPa and in atmospheres containing 0, 1, 3, 5, 10 and 21 % oxygen. Values correspond to the mean values given in Fig. 3. Correlation coefficients of the regression lines were 0n95 (#) and 0n98 ($).
50
40 4C and 4C/2C (%)
21%
30
20
10
0
5
10 15 Oxygen (%)
20
F. 3. Effects of oxygen tension during priming for 7 d at 25 mC with a PEG solution at k1n0 MPa on the subsequent germination rate at 15 mC (expressed as the time to obtain 50 % germination : T ) (A) and &! on the percentage of 4C nuclei (#) and the 4C\2C ratio ($) (B). Means of three (flow cytometry) or four measurements (T ). Vertical &! bars denote the largest s.d. The values of T , 4C and 4C\2C for control &! unprimed seeds were 130p10 h, 7n0p2n0 % and 7n7p1n8 %, respectively (Table 1).
k1n0 MPa, respectively, whereas it reached only 15 or 12 % when priming was performed at k1n5 MPa. In the presence of PEG at k2n0 MPa, the 4C\2C ratio remained at the same low value (about 7 %) as in the control, although the subsequent germination rate was improved.
higher temperature during priming (up to 25–30 mC) reduced T (Fig. 1 A). T was 19 h when priming was carried out at &! &! 25 or 30 mC compared to 130 h for control, unprimed seeds (Table 1). Priming became less efficient at 35 mC. Figure 1 B shows that there was a positive linear relationship between the temperature during priming up to 25 mC and the percentage of 4C nuclei and the 4C\2C ratio, and a negative linear relationship between 25 and 35 mC. Maximum rates of DNA replication were seen at 25 mC ; at this temperature the amount of 4C nuclei and the 4C\2C ratio reached about 29 and 45 %, respectively, after 7 d of priming at k1n0 MPa. At 10 mC only 16 % of the nuclei gave 4C signals and the 4C\2C ratio was only about 20 %. At 35 mC the percentage of 4C nuclei (8 %) and the 4C\2C ratio (8n5 %) remained similar to those measured in unprimed dry seeds (7n0 and 7n7 %, respectively ; Table 1). For temperatures during priming up to 25 mC, a positive correlation existed between the efficiency of the treatment, evaluated by the reciprocal of time to obtain 50 % germination (1\T ) at 15 mC, and the frequency of 4C nuclei &! or the 4C\2C values (Fig. 2). Such a correlation did not exist when priming was performed at higher temperatures. Thus, priming at 35 mC had almost no effect on DNA synthesis while it enhanced the subsequent germination at 15 mC (Fig. 1 A).
Effects of temperature during priming The stimulatory effect of priming (7 d of treatment at k1n0 MPa PEG) on germination and on DNA replication depended on the temperature during treatment (Fig. 1). A
Effects of oxygen tension during priming Priming for 7 d at 25 mC in the complete absence of oxygen did not enhance subsequent germination (Fig. 3 A) ;
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
249
T 2. Effects of NaN concentration during priming for 7 d at 25 mC with a PEG solution at k1n0 MPa on the subsequent $ germination rate at 15 mC, expressed as the time to obtain 50 % germination (T ), the percentages of 2C and 4C nuclei of the &! radicle tips, and the 4C\2C ratio NaN ()
T (h)
2C (%)
4C (%)
(4C\2C)i100
0 10−& 10−% 2i10−% 5i10−% 10−$
19p4 21p4 27p5 39p6 235p15 285p20
63n6p3n8 61n9p1n7 71n6p6n3 82n0p3n2 90n5p2n2 92n9p2n2
28n8p1n3 29n6p1n0 21n0p5n0 12n7p1n8 4n5p0n4 6n4p1n0
45n3p4n8 47n8p0n6 29n3p9n0 15n5p2n7 5n0p0n6 6n9p4n7
$
&!
Data are means of three (flow cytometry) or four measurements (T )ps.d. Values of T , 4C and 4C\2C for control unprimed seeds were &! &! 130p10 h, 7n0p2n0 % and 7n7p1n8 %, respectively (Table 1).
10–5 M
50
0M 4C and 4C/2C (%)
40 10–4 M 30
–5
10
M 0M
2.10–4 M
20
10
5·10 0
–4
2·10
10–3 M –4
0·01
10–4 M M
M 0·02
0·03 0·04 1/T50 (h–1)
0·05
0·06
F. 5. Relationships between the percentages of 4C nuclei (#) or the values of 4C\2C ratio ($) and the subsequent germination rate at 15 mC expressed as the reciprocal of time to obtain 50 % germination (T ). Seeds were primed for 7 d at 25 mC with solutions of PEG at &! k1n0 MPa containing 0, 10−&, 10−%, 2i10−%, 5i10−% and 10−$ NaN . $ Values correspond to the mean values given in Table 2. Correlation coefficients of the regression lines were 0n97 (#) and 0n98 ($).
Effects of priming in the presence of NaN $ Table 2 shows the results obtained with seeds primed for 7 d at 25 mC in k1n0 MPa PEG containing NaN in $ concentrations ranging from 10−& to 10−$ . At concentrations of 10−% and 2i10−% , NaN negatively affected the $ stimulatory effect of priming on subsequent germination at 15 mC and also DNA replication. In the presence of high concentrations of NaN (5i10−% and 10−$ ), most nuclei of $ the radicle tips were maintained in the cell cycle with 2C DNA levels and the priming treatment had no stimulatory effect on subsequent germination. Such a treatment with high concentrations of NaN even retarded subsequent $ germination, since T was about twice as long for seeds &! primed in the presence of 5i10−% and 10−$ NaN $ compared to that of control unprimed seeds (130p10 h ; Table 1). For concentrations of NaN which did not retard $ subsequent germination, e.g. concentrations lower than 5i10−% , a positive linear relationship existed between the 4C or 4C\2C values and the rate of germination at 15 mC, expressed as 1\T (Fig. 5). This correlation was comparable &! to that shown in Fig. 4 concerning the effects of oxygen concentration during priming.
DISCUSSION AND CONCLUSION the mean value of T (134 h) was similar to that obtained &! with unprimed seeds (130 h ; Table 1). Priming required more than 3–5 % oxygen to be effective. Its improving effect increased over the range of oxygen concentrations used in this investigation and was maximal at 21 % oxygen. Levels of oxygen beyond 21 % were not included in the study, but may have had similar\enhanced effects on T values. &! DNA replication was induced during priming only when the treatment was carried out in atmospheres containing more than 5 % oxygen, and it was higher in the air than in 10 % oxygen (Fig. 3 B). Moreover, a linear relationship was observed between the frequency of 4C nuclei or the 4C\2C ratio and the rate of germination at 15 mC (expressed as 1\T ) with seeds primed in various oxygen tensions (Fig. 4). &! Similar results were obtained with seeds primed at 15 mC in the same oxygen concentrations (data not shown).
As previously shown by Bino et al. (1993) and Lanteri et al. (1994), the majority of the nuclei (90n7 %) of the radicle tips of dry mature tomato seeds are arrested in the cell cycle with 2C DNA levels and a low frequency with 4C DNA levels (Table 1). Arrest of some of the nuclei with 4C DNA levels has also been observed with Castanea satia Miller and Raphanus satius L. seeds (Bino et al., 1993). In other species, only 2C signals were found in the embryo radicle tips (Cichorium endia L., Lactuca satia L., Solanum melongena L., Fagus sylatica L. and Pinus nigra J. F. Arnold) while 4C and also 8C signals were observed in radicle cells of Phaseolus ulgaris L. and Spinacia oleracea L. seeds (Bino et al., 1993). Liu et al. (1997) have shown that the arrest of cells in the tomato radicle tip occurs between 30 and 45 d after pollination. In the present study, with ‘ Elko ’ tomato seeds, flow
250
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
cytometric determination of nuclear DNA content showed that osmopriming resulted in replication of DNA in the radicle tip. The amounts of 4C nuclei reached 22n6 and 28n8 % after priming at 25 mC and k1n0 MPa for 3 and 7 d, respectively (Table 1). A comparable induction of DNA replication has also been observed during osmopriming of seeds of other tomato cultivars (‘ Agata ’ and ‘ San Marzano ’) and of pepper (Lanteri et al., 1993, 1994) and during presowing imbibition of sugarbeet seeds (Redfearn and Osborne, 1997). In osmoprimed leek seeds, DNA synthesis would result from both DNA mitochondrial synthesis and repair type synthesis (Ashraf and Bray, 1993). Induction of DNA replication during priming depends on the species, the cultivar and the seed batch (Lanteri et al., 1994), and on the conditions of the treatment. Our results showed that the advancement in DNA synthesis was inversely related to the water potential of the osmoticum (Table 1). In particular, the percentage of 4C nuclei remained close to the initial value measured in dry unprimed seeds (7 %) during 7 d of priming carried out at 25 mC and k2n0 MPa, i.e. when the seed moisture content was less than about 68 % (d. wt basis). Osmotic inhibition of DNA replication has also been observed with pepper seeds, when primed at 20 mC and k1n5 MPa (Lanteri et al., 1994). In tomato seeds, the efficiency of priming was strongly influenced by the temperature of the treatment and was optimal at 25–30 mC (Fig. 1 A). In addition, a positive linear relationship existed between temperature of priming up to 25 mC and the 4C signals or the 4C\2C ratio, and a negative linear relationship at higher temperatures (Fig. 1 B). Similar relationships were demonstrated between (1) the rate of germination (expressed as the reciprocal of time to obtain 50 % germination : 1\T ) of unprimed tomato seeds and &! temperature of germination (O$ zbingo$ l et al., 1998) ; and (2) the rate of germination (also expressed as 1\T ) of primed &! seeds and the temperature of the treatment (O$ zbingo$ l et al., 1998). To affect germination of tomato seeds, the priming treatment required at least 5 % oxygen in the atmosphere (Fig. 3 A), indicating that respiration was necessary for processes associated with priming (Bray, 1995). Similar oxygen concentrations were also required for DNA replication during priming (Fig. 3 B) and for germination of unprimed seeds (O$ zbingo$ l et al., 1998). The requirement for energy metabolism during priming was also demonstrated by the treatments with NaN (Table 2). Inefficiency of $ priming in low oxygen tensions or in the presence of high concentrations of NaN (5i10−% and 10−$ ) was asso$ ciated in both cases with a reduced energy charge (data not shown). As in pepper seeds (Lanteri et al., 1993, 1994), a close positive relationship was observed between the percentage of 4C nuclei in the radicle tip after priming, or the 4C\2C ratio and the beneficial effect of the osmotic treatment in tomato seeds (Figs 2, 4 and 5). However, there were some limits to this relationship, since osmopriming at too high a temperature (35 mC) (Fig. 1) or with a solution of PEG at a low water potential (k2n0 MPa) (Table 1) improved subsequent germination without generating an increase in 4C signals. These results, which agree with those obtained
by Saracco et al. (1995) with pepper seeds primed for 6 d at 20 mC with a solution of PEG at k1n5 MPa, show that improvement of seed germinability by priming is not always associated with the onset of DNA replication. A C K N O W L E D G E M E N TS Jan H. W. Bergervoet and Jaap G. van Pijlen are acknowledged for their technical advice with the flow cytometric measurements. Part of the research presented in this paper has been made possible through support of the Commission of the European Communities within the Concerted Action project ‘ Integration of physiological and molecular disciplines in seed quality analysis ’ (AIR3-CT94-1863). However, the content of this paper is the sole responsibility of the authors and in no way represents the views of the Commission of the European Communities, Division for Coordination of Agricultural Research, DGVI.F.II-3 or its services. LITERATURE CITED Ashraf M, Bray CM. 1993. DNA synthesis in osmoprimed leek (Allium porrum L.) seeds and evidence for repair and replication. Seed Science Research 3 : 15–23. Bailly C, Benamar A, Corbineau F, Co# me D. 1997. Changes in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds during accelerated ageing and subsequent priming. In : Ellis RH, Black M, Murdoch AJ, Hong TD, eds. Basic and applied aspects of seed biology. Dordrecht : Kluwer Academic Publishers, 665–672. Bino RJ, De Vries JN, Kraak HL, Van Pijlen JG. 1992. Flow cytometric determination of nuclear DNA replication stages in tomato seeds during priming and germination. Annals of Botany 69 : 231–236. Bino RJ, Lanteri S, Verhoeven HA, Kraak HL. 1993. Flow cytometric determination of nuclear replication stages in seed tissues. Annals of Botany 72 : 181–187. Bray CM. 1995. Biochemical processes during the osmopriming of seeds. In : Kigel J, Galili G, eds. Seed deelopment and germination. New York : Marcel Dekker, 767–789. Bray CM, Davison PA, Ashraf M, Taylor RM. 1989. Biochemical changes during priming of leek seeds. Annals of Botany 63 : 185–193. Brocklehurst PA, Dearman J. 1983. Interaction between seed priming treatments and nine seed lots of carrot, celery and onion. I. Laboratory germination. Annals of Applied Biology 102 : 577–584. Chojnowski M, Corbineau F, Co# me D. 1997. Physiological and biochemical changes induced in sunflower seeds by osmopriming and subsequent drying, storage and aging. Seed Science Research 7 : 323–331. Co# me D, Tissaoui T. 1968. Induction d’une dormance embryonnaire secondaire chez le pommier (Pirus malus L.) par des atmosphe' res tre' s appauvries en oxyge' ne. Comptes Rendus de l’AcadeT mie des Sciences, Paris 266 : Se! rie D, 477–479. Coolbear P, Grierson D. 1979. Studies on the changes in the major nucleic acid components of tomato seeds (Lycopersicon esculentum Mill.) resulting from osmotic presowing treatment. Journal of Experimental Botany 30 : 1153–1162. Corbineau F, Co# me D. 1990. Effects of priming on the germination of Valerianella olitoria seeds in relation with temperature and oxygen. Acta Horticulturae 267 : 191–197. Corbineau F, Picard MA, Co# me D. 1993. Germinability of some vegetable seeds in relation to temperature and oxygen. In : Co# me D, Corbineau F, eds. Fourth international workshop on seeds. Basic and applied aspects of seed biology, Vol. 3. Paris : ASFIS, 1027–1032. Corbineau F, Picard MA, Co# me D. 1994. Germinability of leek seeds and its improvement by osmopriming. Acta Horticulturae 371 : 45–52. Davison PA, Bray CM. 1991. Protein synthesis during osmopriming of leek (Allium porrum L.) seeds. Seed Science Research 1 : 29–35.
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds De Castro RD, Zheng X, Bergervoet JHW, De Vos CHR, Bino RJ. 1995. β-tubulin accumulation and DNA replication in imbibing tomato seeds. Plant Physiology 109 : 499–504. Dell’Aquila A, Bewley JD. 1989. Protein synthesis in the axes of polyethylene glycol treated pea seeds and during subsequent germination. Journal of Experimental Botany 40 : 1001–1007. Guedes AC, Cantliffe DJ. 1980. Germination of lettuce seeds at high temperature after seed priming. Journal of American Society for Horticultural Science 105 : 777–781. Halpin-Ingham B, Sundstrom FJ. 1992. Pepper seed water content, germination response and respiration following priming treatments. Seed Science and Technology 20 : 589–596. Heydecker W, Higgins J, Turner YJ. 1975. Invigoration of seeds ? Seed Science and Technology 3 : 881–888. Lanteri S, Kraak HL, De Vos CHR, Bino RJ. 1993. Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper (Capsicum annuum). Physiologia Plantarum 89 : 433–440. Lanteri S, Saracco F, Kraak HL, Bino RJ. 1994. The effects of priming on nuclear replication activity and germination of pepper (Capsicum annuum) and tomato (Lycopersicon esculentum) seeds. Seed Science Research 4 : 81–87.
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Liu Y, Hilhorst HWM, Groot SPG, Bino RJ. 1997. Amounts of nuclear DNA and internal morphology of gibberellin- and abscisic aciddeficient tomato (Lycopersicon esculentum Mill.) seeds during maturation, imbibition and germination. Annals of Botany 79 : 161–168. Michel BE, Kaufmann, MR. 1973. The osmotic potential of polyethylene glycol 6000. Plant Physiology 51 : 914–916. O$ zbingo$ l N, Corbineau F, Co# me D. 1998. Responses of tomato seeds to osmoconditioning as related to temperature and oxygen. Seed Science Research 8 : 377–384. Redfearn M, Osborne DJ. 1997. Effects of advancement on nucleic acids in sugarbeet (Beta ulgaris) seeds. Seed Science Research 7 : 261–267. Saracco F, Bino RJ, Bergervoet JHW, Lanteri S. 1995. Influence of priming-induced nuclear replication activity on storability of pepper (Capsicum annuum L.) seed. Seed Science Research 5 : 25–29. Smok MA, Chojnowski M, Corbineau F, Co# me D. 1993. Effects of osmotic treatment on sunflower seed germination in relation with temperature and oxygen. In : Co# me D, Corbineau F, eds. Fourth international workshop on seeds. Basic and applied aspects of seed biology, Vol. 3. Paris : ASFIS, 1033–1038.
Activation of the Cell Cycle in Tomato (Lycopersicon esculentum Mill.) Seeds during Osmoconditioning as Related to Temperature and Oxygen N. O$ Z B I N G O$ L*, F. C O R B I N E A U*†, S. P. C. G R O OT‡, R. J. B I N O‡ and D. C O# M E* * UniersiteT Pierre et Marie Curie, Laboratoire de Physiologie VeT geT tale AppliqueT e, Tour 53, 1er eT tage, 4 Place Jussieu, 75252 Paris CeT dex 05, France, ‡ CPRO-DLO, P.O. Box 16, 6700 AA Wageningen, The Netherlands Received : 8 March 1999
Returned for revision : 14 April 1999
Accepted : 4 May 1999
Using flow cytometric analyses of the nuclear DNA content, we studied the effects of various conditions of osmopriming on the activation of the cell cycle in embryo root tips of tomato (Lycopersicon esculentum ‘ Elko ’) seeds. In dry untreated seeds, 90n7 % of the nuclei revealed 2C signals. Priming of seeds in polyethylene glycol-8000 (PEG) improved the germination rate of seeds transferred onto water at 15 mC. This was associated with an increase in 4C signals when priming was carried out at k1n0 and k1n5 MPa. Priming at k2n0 MPa enhanced subsequent germination but had no effect on DNA replication. For temperatures during priming up to 25 mC, a positive linear correlation existed between the efficiency of the treatment, evaluated by the reciprocal of time to obtain 50 % germination at 15 mC, and the frequency of 4C nuclei or the 4C\2C values. Such a correlation did not exist when priming was performed at higher temperatures. At least 5 % oxygen in the atmosphere was required during priming for the induction of DNA replication and for the enhancement of subsequent germination. In the presence of 5i10−% and 10−$ NaN during priming, most of the cells were maintained with 2C DNA levels and the treatment had no $ stimulatory effect on germination. The results show a positive linear relationship between the frequency of 4C DNA nuclei or the 4C\2C ratio and the improving effect of priming. However, in suboptimal conditions of priming (k2n0 MPa or temperatures higher than 25 mC), the improvement of seed germination was not associated with the onset of DNA replication. # 1999 Annals of Botany Company Key words : Cell cycle, germination, osmopriming, oxygen, temperature, Lycopersicon esculentum, tomato.
INTRODUCTION Osmopriming consists of imbibing seeds in an osmotic solution at low water potential in order to control their water uptake and prevent radicle protrusion. Such treatment improves the germination of seeds of numerous species (Heydecker, Higgins and Turner, 1975 ; Guedes and Cantliffe, 1980 ; Brocklehurst and Dearman, 1983 ; Corbineau, Picard and Co# me, 1993 ; Bray, 1995). Primed seeds germinate in a wider range of temperatures (Bray, 1995) and are less sensitive to oxygen deprivation (Corbineau and Co# me, 1990 ; Corbineau et al., 1993 ; Smok et al., 1993 ; Corbineau, Picard and Co# me, 1994) than unprimed ones. The beneficial effect of priming has been associated with various biochemical, cellular and molecular events including synthesis of RNA and proteins (Bray et al., 1989 ; Dell’Aquila and Bewley, 1989 ; Davison and Bray, 1991 ; Bray, 1995). Priming is also known to enhance respiratory activity of seeds (Halpin-Ingham and Sundstrom, 1992 ; Chojnowski, Corbineau and Co# me, 1997) and, when applied to aged seeds, restores activities of enzymes involved in the cell detoxifying mechanisms such as superoxide dismutase, catalase and glutathione reductase (Bailly et al., 1997). Studies on the effects of osmopriming on DNA replication † For correspondence. Fax j33 1 44 27 59 27, e-mail corbi!ccr.jussieu.fr
0305-7364\99\080245j07 $30.00\0
have given inconsistent results. Only low DNA synthesis was found during priming in leek (Allium porrum L.) (Bray et al., 1989 ; Ashraf and Bray, 1993) and tomato (Lycopersicon esculentum Miller) (Coolbear and Grierson, 1979) seeds. In contrast, studies using flow cytometry have demonstrated that DNA replication was initiated in the radicle tip cells during osmopriming of seeds of tomato (Bino et al., 1992 ; Lanteri et al., 1994) and pepper (Capsicum annuum L.) (Lanteri et al., 1993, 1994 ; Saracco et al., 1995). An increase in 4C DNA content was also observed in sugarbeet (Beta ulgaris L.) seeds (Redfearn and Osborne, 1997). Induction of DNA replication has also been associated with an accumulation of β-tubulin (De Castro et al., 1995). In pepper and tomato seeds, a positive correlation was found between the induction of DNA replication, measured as the increase in 4C nuclei, and the efficiency of the osmotic treatment (Lanteri et al., 1994). The percentage of 4C nuclei started to increase after 3 d of priming in the radicle tips of tomato embryos (Bino et al., 1992) and after 10 d in pepper embryos (Lanteri et al., 1993). A reduction of the polyethylene glycol concentration used for the priming treatment resulted in a greater proportion of cells with 4C DNA content (Lanteri et al., 1994 ; Saracco et al., 1995). Nevertheless, Saracco et al. (1995) found that priming could improve germination of pepper seeds without an increase in 4C nuclei. The aims of the present work were : (1) to investigate # 1999 Annals of Botany Company
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
246
T 1. Effects of 3 or 7 d of priming at 25 mC with PEG solutions at k1n0, k1n5 or k2n0 MPa on the seed moisture content (dry weight basis) before redrying, the subsequent germination rate at 15 mC, expressed as the time to obtain 50 % germination (T ), the percentages of 2C and 4C nuclei of the radicle tips, and the 4C\2C ratio &! Conditions of priming Duration (d) 0 (control) 3
7
Water potential (MPa)
Moisture content (% d. wt)
T (h)
2C (%)
4C (%)
(4C\2C) i100
— k1n0 k1n5 k2n0 k1n0 k1n5 k2n0
9n4p0n4 77n3p4n2 70n2p4n3 64n8p8n2 76n0p2n4 68n8p1n7 67n6p3n8
130p10 50p1 52p2 70p5 19p1 40p2 48p4
90n7p0n4 77n2p1n0 86n9p0n6 93n8p0n3 63n6p3n8 89n0p0n2 93n0p0n9
7n0p2n0 22n6p0n8 13n0p0n5 6n2p0n5 28n8p1n3 10n9p0n5 6n8p0n2
7n7p1n8 29n3p1n3 15n0p0n2 6n6p0n6 45n3p4n8 12n2p0n8 7n3p0n3
&!
Control seeds were not primed. Data are means of three (flow cytometry and moisture content) or four measurements (T )ps.d.
&!
effects of conditions of priming (water potential, oxygen and temperature) on tomato seed germination and DNA replication ; and (2) to determine whether the beneficial effect of priming is correlated with an increase in 4C nuclei. MATERIALS AND METHODS Plant material Experiments were carried out with seeds of tomato (Lycopersicon esculentum Mill., ‘ Elko ’) supplied by Clause Semences (France). Seeds were stored dry in the open air at 20 mC and 55 % relative humidity before the experiments started. Osmopriming treatments Seeds were primed for 7 d on a layer of cotton wool imbibed with polyethylene glycol-8000 (PEG) solutions at k1n0, k1n5 and k2n0 MPa. The treatments were carried out at temperatures ranging from 10 to 35 mC (p0n5 mC). Concentrations of PEG solutions were calculated according to Michel and Kaufmann (1973). Priming of seeds in various oxygen tensions (mixtures of nitrogen and oxygen) was performed for 7 d at 25 mC—the optimal temperature for priming of tomato seeds (O$ zbingo$ l, Corbineau and Co# me, 1998)—according to the procedure developed by Co# me and Tissaoui (1968). Gas mixtures were obtained through capillary tubes by mixing compressed air and pure nitrogen. The atmospheres thus obtained were passed continuously through germination chambers at a constant flow rate (4 l h−"). They contained 0 (pure nitrogen), 1, 3, 5, 10 and 21 % oxygen (air). In order to study whether a particular level of respiratory activity was necessary to achieve priming, seeds were soaked for 7 d at 25 mC in polyethylene glycol solutions at k1n0 MPa containing various concentrations of the respiratory inhibitor NaN ranging from 10−& to 10−$ . $ After the osmotic treatments, seeds were rinsed for 30 s with distilled water and were dried for 3 d in the open air at
20 mC and 55 % relative humidity. After redrying, the moisture content of primed seeds was similar (8–9 % d. wt basis) to that of control untreated seeds. Dry weight was obtained by oven drying seeds at 105 mC for 3 d.
Germination assays Germination ability was tested in samples of 100 seeds placed in 9 cm diameter Petri dishes (25 seeds per dish, four replicates) on a layer of cotton wool moistened with distilled water. Germination assays were carried out at 15 mC in darkness. A seed was regarded as germinated when the radicle protruded through the seed coat. Germination counts were made daily up to 15 d. Results were expressed as the time to reach 50 % germination (T ) or the reciprocal &! of T (1\T ). Results presented are the means of the results &! &! obtained in four replicatesps.d.
Flow cytometry Amounts of nuclear DNA were quantified using radicle tips of embryos isolated from control unprimed seeds and from seeds primed for 7 d in various conditions as specified earlier. Radicle tips (1 mm) were dissected from seeds, dried for 2 d at 20 mC in the presence of silica gel and stored at k30 mC for later use. Embryo radicle tips were then chopped with a razor blade in 500 µl of nucleus-isolation buffer containing 0n2 mannitol, 10 m 2(-morpholino) ethanesulphonic acid, 10 m NaCl, 10 m KCl, 10 m spermine tetrahydrochloride, 2n5 m ethylenediamine-tetraacetic acid, 2n5 m dithiothreitol, 0n05 % v\v triton X-100 and 0n05 % NaN at pH 5n8 as previously described by Bino et al. $ (1993). The mixture and additional 500 µl nucleus-isolation buffer were sieved through 88 µm nylon mesh into the flowcytometer test tubes. Twenty µl of 1 mg ml−" propidium iodide solution (Molecular Probes, Eugene, OR, USA) were then added to allow measurement of amounts of nuclear DNA by fluorescence. For each sample, five radicle tips, representing about 5000 to 10 000 nuclei, were used and
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds flow-cytometric determinations were made at least in duplicate. The DNA content of isolated nuclei was measured about 10 min after sample staining, using a flow cytometer (Coulter Corp., Miami, FL, USA, Epics XL-MCL model). Excitation of DNA-bound propidium iodide was performed by a 488 nm Argon Ion Laser, and fluorescence was detected over the range 605–635 nm. MultiCycle for Windows Cell Cycle Analysis software version 3n0 (Phoenix, Flow System Inc., San Diego, CA, USA) was used for curve fitting of fluorescence frequency distributions. The DNA amount is
A 100
T50 (h)
80
60
40
20
0
5
10
15
20
25
30
35
40
247
proportional to the fluorescent signal and is expressed as an arbitrary C value in which the 1C value represents the DNA content of the unreplicated haploid chromosome complement. Results presented are the means of the percentages of nuclei with 2C DNA levels and those with 4C DNA levels.
RESULTS Effects of priming at arious water potentials Effects on seed germination of priming for 3 and 7 d at 25 mC with PEG solutions at k1n0, k1n5 and k2n0 MPa are presented in Table 1. The moisture content of the seeds increased with higher water potentials. No seeds germinated during the osmotic treatments (data not shown). In all cases, primed seeds germinated faster and the time to obtain 50 % germination (T ) was decreased compared to that of &! unprimed seeds. The improvement of germination was higher after 7 d of priming than after 3 d, and a water potential of k1n0 MPa was the most efficient. For this reason, in all the following experiments seeds were primed for 7 d with a PEG solution at k1n0 MPa. Table 1 also shows the effects of priming on nuclear DNA content of radicle tip cells. In dry unprimed seeds, 90n7 % of the nuclei gave 2C signals, indicating that the majority of cells were arrested at the G phase of the cell cycle. Priming " with PEG at k1n5 and k1n0 MPa resulted in an increase of 4C signals, which was more marked with the highest water potential. However, priming with PEG at k2n0 MPa had no effect on DNA replication. The 4C\2C ratio increased up to about 29 and 45 % after 3 and 7 d of priming at
B 50 50 25° 40 30
4C and 4C/2C (%)
4C and 4C/2C (%)
40
20
20° 25°
30 20° 15° 20
30°
10
15°
10° 35°
10 0
5
10 15 20 30 25 Priming temperature (°C)
35
35°
40
F. 1. Effects of temperature during priming for 7 d with a PEG solution at k1n0 MPa on the subsequent germination rate at 15 mC (expressed as the time to obtain 50 % germination : T ) (A) and on the &! percentage of 4C nuclei (#) and the 4C\2C ratio ($) (B). Means of three (flow cytometry) or four measurements (T ). Vertical bars denote &! the largest s.d. Correlation coefficients of the regression lines in B were between 0n90 and 0n98. The values of T , 4C and 4C\2C for control &! unprimed seeds were 130p10 h, 7n0p2n0 % and 7n7p1n8 %, respectively (Table 1).
30°
10°
0
0·01
0·02
0·03 0·04 1/T50 (h–1)
0·05
0·06
F. 2. Relationships between the percentages of 4C nuclei (#) or the values of the 4C\2C ratio ($) and the subsequent germination rate at 15 mC, expressed as the reciprocal of time to obtain 50 % germination (T ). Seeds were primed for 7 d at 10, 15, 20, 25, 30 and 35 mC with a &! PEG solution at k1n0 MPa. Values correspond to the mean values given in Fig. 1.
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
248 A
50
150
21% 125 4C and 4C/2C (%)
40
T50 (h)
100
75
30
10%
20 10%
50 10
25
0
5
10
15
20
B
0
3% 0%
5% 1% 0·01
0·02
0·03 0·04 1/T50 (h–1)
0·05
0·06
F. 4. Relationships between the percentages of 4C nuclei (#) or the values of the 4C\2C ratio ($) and the subsequent germination rate at 15 mC expressed as the reciprocal of time to obtain 50 % germination (T ). Seeds were primed for 7 d at 25 mC with a PEG solution at &! k1n0 MPa and in atmospheres containing 0, 1, 3, 5, 10 and 21 % oxygen. Values correspond to the mean values given in Fig. 3. Correlation coefficients of the regression lines were 0n95 (#) and 0n98 ($).
50
40 4C and 4C/2C (%)
21%
30
20
10
0
5
10 15 Oxygen (%)
20
F. 3. Effects of oxygen tension during priming for 7 d at 25 mC with a PEG solution at k1n0 MPa on the subsequent germination rate at 15 mC (expressed as the time to obtain 50 % germination : T ) (A) and &! on the percentage of 4C nuclei (#) and the 4C\2C ratio ($) (B). Means of three (flow cytometry) or four measurements (T ). Vertical &! bars denote the largest s.d. The values of T , 4C and 4C\2C for control &! unprimed seeds were 130p10 h, 7n0p2n0 % and 7n7p1n8 %, respectively (Table 1).
k1n0 MPa, respectively, whereas it reached only 15 or 12 % when priming was performed at k1n5 MPa. In the presence of PEG at k2n0 MPa, the 4C\2C ratio remained at the same low value (about 7 %) as in the control, although the subsequent germination rate was improved.
higher temperature during priming (up to 25–30 mC) reduced T (Fig. 1 A). T was 19 h when priming was carried out at &! &! 25 or 30 mC compared to 130 h for control, unprimed seeds (Table 1). Priming became less efficient at 35 mC. Figure 1 B shows that there was a positive linear relationship between the temperature during priming up to 25 mC and the percentage of 4C nuclei and the 4C\2C ratio, and a negative linear relationship between 25 and 35 mC. Maximum rates of DNA replication were seen at 25 mC ; at this temperature the amount of 4C nuclei and the 4C\2C ratio reached about 29 and 45 %, respectively, after 7 d of priming at k1n0 MPa. At 10 mC only 16 % of the nuclei gave 4C signals and the 4C\2C ratio was only about 20 %. At 35 mC the percentage of 4C nuclei (8 %) and the 4C\2C ratio (8n5 %) remained similar to those measured in unprimed dry seeds (7n0 and 7n7 %, respectively ; Table 1). For temperatures during priming up to 25 mC, a positive correlation existed between the efficiency of the treatment, evaluated by the reciprocal of time to obtain 50 % germination (1\T ) at 15 mC, and the frequency of 4C nuclei &! or the 4C\2C values (Fig. 2). Such a correlation did not exist when priming was performed at higher temperatures. Thus, priming at 35 mC had almost no effect on DNA synthesis while it enhanced the subsequent germination at 15 mC (Fig. 1 A).
Effects of temperature during priming The stimulatory effect of priming (7 d of treatment at k1n0 MPa PEG) on germination and on DNA replication depended on the temperature during treatment (Fig. 1). A
Effects of oxygen tension during priming Priming for 7 d at 25 mC in the complete absence of oxygen did not enhance subsequent germination (Fig. 3 A) ;
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
249
T 2. Effects of NaN concentration during priming for 7 d at 25 mC with a PEG solution at k1n0 MPa on the subsequent $ germination rate at 15 mC, expressed as the time to obtain 50 % germination (T ), the percentages of 2C and 4C nuclei of the &! radicle tips, and the 4C\2C ratio NaN ()
T (h)
2C (%)
4C (%)
(4C\2C)i100
0 10−& 10−% 2i10−% 5i10−% 10−$
19p4 21p4 27p5 39p6 235p15 285p20
63n6p3n8 61n9p1n7 71n6p6n3 82n0p3n2 90n5p2n2 92n9p2n2
28n8p1n3 29n6p1n0 21n0p5n0 12n7p1n8 4n5p0n4 6n4p1n0
45n3p4n8 47n8p0n6 29n3p9n0 15n5p2n7 5n0p0n6 6n9p4n7
$
&!
Data are means of three (flow cytometry) or four measurements (T )ps.d. Values of T , 4C and 4C\2C for control unprimed seeds were &! &! 130p10 h, 7n0p2n0 % and 7n7p1n8 %, respectively (Table 1).
10–5 M
50
0M 4C and 4C/2C (%)
40 10–4 M 30
–5
10
M 0M
2.10–4 M
20
10
5·10 0
–4
2·10
10–3 M –4
0·01
10–4 M M
M 0·02
0·03 0·04 1/T50 (h–1)
0·05
0·06
F. 5. Relationships between the percentages of 4C nuclei (#) or the values of 4C\2C ratio ($) and the subsequent germination rate at 15 mC expressed as the reciprocal of time to obtain 50 % germination (T ). Seeds were primed for 7 d at 25 mC with solutions of PEG at &! k1n0 MPa containing 0, 10−&, 10−%, 2i10−%, 5i10−% and 10−$ NaN . $ Values correspond to the mean values given in Table 2. Correlation coefficients of the regression lines were 0n97 (#) and 0n98 ($).
Effects of priming in the presence of NaN $ Table 2 shows the results obtained with seeds primed for 7 d at 25 mC in k1n0 MPa PEG containing NaN in $ concentrations ranging from 10−& to 10−$ . At concentrations of 10−% and 2i10−% , NaN negatively affected the $ stimulatory effect of priming on subsequent germination at 15 mC and also DNA replication. In the presence of high concentrations of NaN (5i10−% and 10−$ ), most nuclei of $ the radicle tips were maintained in the cell cycle with 2C DNA levels and the priming treatment had no stimulatory effect on subsequent germination. Such a treatment with high concentrations of NaN even retarded subsequent $ germination, since T was about twice as long for seeds &! primed in the presence of 5i10−% and 10−$ NaN $ compared to that of control unprimed seeds (130p10 h ; Table 1). For concentrations of NaN which did not retard $ subsequent germination, e.g. concentrations lower than 5i10−% , a positive linear relationship existed between the 4C or 4C\2C values and the rate of germination at 15 mC, expressed as 1\T (Fig. 5). This correlation was comparable &! to that shown in Fig. 4 concerning the effects of oxygen concentration during priming.
DISCUSSION AND CONCLUSION the mean value of T (134 h) was similar to that obtained &! with unprimed seeds (130 h ; Table 1). Priming required more than 3–5 % oxygen to be effective. Its improving effect increased over the range of oxygen concentrations used in this investigation and was maximal at 21 % oxygen. Levels of oxygen beyond 21 % were not included in the study, but may have had similar\enhanced effects on T values. &! DNA replication was induced during priming only when the treatment was carried out in atmospheres containing more than 5 % oxygen, and it was higher in the air than in 10 % oxygen (Fig. 3 B). Moreover, a linear relationship was observed between the frequency of 4C nuclei or the 4C\2C ratio and the rate of germination at 15 mC (expressed as 1\T ) with seeds primed in various oxygen tensions (Fig. 4). &! Similar results were obtained with seeds primed at 15 mC in the same oxygen concentrations (data not shown).
As previously shown by Bino et al. (1993) and Lanteri et al. (1994), the majority of the nuclei (90n7 %) of the radicle tips of dry mature tomato seeds are arrested in the cell cycle with 2C DNA levels and a low frequency with 4C DNA levels (Table 1). Arrest of some of the nuclei with 4C DNA levels has also been observed with Castanea satia Miller and Raphanus satius L. seeds (Bino et al., 1993). In other species, only 2C signals were found in the embryo radicle tips (Cichorium endia L., Lactuca satia L., Solanum melongena L., Fagus sylatica L. and Pinus nigra J. F. Arnold) while 4C and also 8C signals were observed in radicle cells of Phaseolus ulgaris L. and Spinacia oleracea L. seeds (Bino et al., 1993). Liu et al. (1997) have shown that the arrest of cells in the tomato radicle tip occurs between 30 and 45 d after pollination. In the present study, with ‘ Elko ’ tomato seeds, flow
250
OW zbingoW l et al.—Cell Cycle in Osmoconditioned Tomato Seeds
cytometric determination of nuclear DNA content showed that osmopriming resulted in replication of DNA in the radicle tip. The amounts of 4C nuclei reached 22n6 and 28n8 % after priming at 25 mC and k1n0 MPa for 3 and 7 d, respectively (Table 1). A comparable induction of DNA replication has also been observed during osmopriming of seeds of other tomato cultivars (‘ Agata ’ and ‘ San Marzano ’) and of pepper (Lanteri et al., 1993, 1994) and during presowing imbibition of sugarbeet seeds (Redfearn and Osborne, 1997). In osmoprimed leek seeds, DNA synthesis would result from both DNA mitochondrial synthesis and repair type synthesis (Ashraf and Bray, 1993). Induction of DNA replication during priming depends on the species, the cultivar and the seed batch (Lanteri et al., 1994), and on the conditions of the treatment. Our results showed that the advancement in DNA synthesis was inversely related to the water potential of the osmoticum (Table 1). In particular, the percentage of 4C nuclei remained close to the initial value measured in dry unprimed seeds (7 %) during 7 d of priming carried out at 25 mC and k2n0 MPa, i.e. when the seed moisture content was less than about 68 % (d. wt basis). Osmotic inhibition of DNA replication has also been observed with pepper seeds, when primed at 20 mC and k1n5 MPa (Lanteri et al., 1994). In tomato seeds, the efficiency of priming was strongly influenced by the temperature of the treatment and was optimal at 25–30 mC (Fig. 1 A). In addition, a positive linear relationship existed between temperature of priming up to 25 mC and the 4C signals or the 4C\2C ratio, and a negative linear relationship at higher temperatures (Fig. 1 B). Similar relationships were demonstrated between (1) the rate of germination (expressed as the reciprocal of time to obtain 50 % germination : 1\T ) of unprimed tomato seeds and &! temperature of germination (O$ zbingo$ l et al., 1998) ; and (2) the rate of germination (also expressed as 1\T ) of primed &! seeds and the temperature of the treatment (O$ zbingo$ l et al., 1998). To affect germination of tomato seeds, the priming treatment required at least 5 % oxygen in the atmosphere (Fig. 3 A), indicating that respiration was necessary for processes associated with priming (Bray, 1995). Similar oxygen concentrations were also required for DNA replication during priming (Fig. 3 B) and for germination of unprimed seeds (O$ zbingo$ l et al., 1998). The requirement for energy metabolism during priming was also demonstrated by the treatments with NaN (Table 2). Inefficiency of $ priming in low oxygen tensions or in the presence of high concentrations of NaN (5i10−% and 10−$ ) was asso$ ciated in both cases with a reduced energy charge (data not shown). As in pepper seeds (Lanteri et al., 1993, 1994), a close positive relationship was observed between the percentage of 4C nuclei in the radicle tip after priming, or the 4C\2C ratio and the beneficial effect of the osmotic treatment in tomato seeds (Figs 2, 4 and 5). However, there were some limits to this relationship, since osmopriming at too high a temperature (35 mC) (Fig. 1) or with a solution of PEG at a low water potential (k2n0 MPa) (Table 1) improved subsequent germination without generating an increase in 4C signals. These results, which agree with those obtained
by Saracco et al. (1995) with pepper seeds primed for 6 d at 20 mC with a solution of PEG at k1n5 MPa, show that improvement of seed germinability by priming is not always associated with the onset of DNA replication. A C K N O W L E D G E M E N TS Jan H. W. Bergervoet and Jaap G. van Pijlen are acknowledged for their technical advice with the flow cytometric measurements. Part of the research presented in this paper has been made possible through support of the Commission of the European Communities within the Concerted Action project ‘ Integration of physiological and molecular disciplines in seed quality analysis ’ (AIR3-CT94-1863). However, the content of this paper is the sole responsibility of the authors and in no way represents the views of the Commission of the European Communities, Division for Coordination of Agricultural Research, DGVI.F.II-3 or its services. LITERATURE CITED Ashraf M, Bray CM. 1993. DNA synthesis in osmoprimed leek (Allium porrum L.) seeds and evidence for repair and replication. Seed Science Research 3 : 15–23. Bailly C, Benamar A, Corbineau F, Co# me D. 1997. Changes in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds during accelerated ageing and subsequent priming. In : Ellis RH, Black M, Murdoch AJ, Hong TD, eds. Basic and applied aspects of seed biology. Dordrecht : Kluwer Academic Publishers, 665–672. Bino RJ, De Vries JN, Kraak HL, Van Pijlen JG. 1992. Flow cytometric determination of nuclear DNA replication stages in tomato seeds during priming and germination. Annals of Botany 69 : 231–236. Bino RJ, Lanteri S, Verhoeven HA, Kraak HL. 1993. Flow cytometric determination of nuclear replication stages in seed tissues. Annals of Botany 72 : 181–187. Bray CM. 1995. Biochemical processes during the osmopriming of seeds. In : Kigel J, Galili G, eds. Seed deelopment and germination. New York : Marcel Dekker, 767–789. Bray CM, Davison PA, Ashraf M, Taylor RM. 1989. Biochemical changes during priming of leek seeds. Annals of Botany 63 : 185–193. Brocklehurst PA, Dearman J. 1983. Interaction between seed priming treatments and nine seed lots of carrot, celery and onion. I. Laboratory germination. Annals of Applied Biology 102 : 577–584. Chojnowski M, Corbineau F, Co# me D. 1997. Physiological and biochemical changes induced in sunflower seeds by osmopriming and subsequent drying, storage and aging. Seed Science Research 7 : 323–331. Co# me D, Tissaoui T. 1968. Induction d’une dormance embryonnaire secondaire chez le pommier (Pirus malus L.) par des atmosphe' res tre' s appauvries en oxyge' ne. Comptes Rendus de l’AcadeT mie des Sciences, Paris 266 : Se! rie D, 477–479. Coolbear P, Grierson D. 1979. Studies on the changes in the major nucleic acid components of tomato seeds (Lycopersicon esculentum Mill.) resulting from osmotic presowing treatment. Journal of Experimental Botany 30 : 1153–1162. Corbineau F, Co# me D. 1990. Effects of priming on the germination of Valerianella olitoria seeds in relation with temperature and oxygen. Acta Horticulturae 267 : 191–197. Corbineau F, Picard MA, Co# me D. 1993. Germinability of some vegetable seeds in relation to temperature and oxygen. In : Co# me D, Corbineau F, eds. Fourth international workshop on seeds. Basic and applied aspects of seed biology, Vol. 3. Paris : ASFIS, 1027–1032. Corbineau F, Picard MA, Co# me D. 1994. Germinability of leek seeds and its improvement by osmopriming. Acta Horticulturae 371 : 45–52. Davison PA, Bray CM. 1991. Protein synthesis during osmopriming of leek (Allium porrum L.) seeds. Seed Science Research 1 : 29–35.
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