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Tree Physiology 20, 615–621
© 2000 Heron Publishing—Victoria, Canada
Influence of temperature on the dynamics of ATP, ADP and
non-adenylic triphosphate nucleotides in vegetative and floral peach
buds during dormancy
M. BONHOMME,1 R. RAGEAU1 and M. GENDRAUD2
1
U.A.Bioclimatologie-PIAF, INRA Domaine de Crouelle, 234 avenue du Brezet, F63039 Clermont-Ferrand Cedex, France
2
U.A.Bioclimatologie-PIAF, Université Blaise Pascal, 24 avenue des Landais, F63177 Aubière, France
Received June 11, 1999
Summary The nucleotides test of endodormancy, which is
based on the capacity of tissues to synthesize ATP and nonadenylic triphosphate nucleotides (NTP), cannot be used for
floral buds, and it is of questionable use for vegetative buds. In
an attempt to find an alternative test, we examined whether the
dormancy state of vegetative and floral buds of trees exposed
to different temperature conditions during the rest period is directly related to their ATP, ADP and NTP concentrations and
ATP/ADP ratio. Once the buds had entered endo- or paradormancy, the nucleotide concentrations and the ATP/ADP ratio were low in the vegetative primordia and very low in the
floral primordia. Only after the action of chilling, when the
buds were considered to have completed the endodormancy
and paradormancy phases, did the nucleotide concentrations
increase, accompanied by a steep rise in ATP/ADP ratio. We
conclude that the ATP/ADP ratio could be used to characterize
the bud dormancy state by comparison with critical values of
1.5 for vegetative primordia and 1.0 for floral primordia.
Keywords: ecodormancy, endodormancy, floral bud, NTP,
paradormancy, peach tree, Prunus persica, vegetative bud.
Introduction
In perennials, the control of bud growth is exerted by environmental factors, mainly temperature, and for some species,
photoperiod (Heide 1993), by correlative influences from
other buds or tissues, or by processes occurring within the bud
itself, more precisely within the meristematic zone. These
three mechanisms of control of bud growth are referred to as
ecodormancy, paradormancy and endodormancy, respectively, in the terminology of Lang et al. (1987).
Endodormancy is released by the action of chilling. The different kinds of dormancy can act simultaneously (Saure 1985,
Champagnat 1989), but the typical sequence of dormancies
during the non-vegetative season (from leaf shedding to
budbreak) is generally paradormancy followed by
endodormancy and then ecodormancy (Fuchigami and
Wisniewski 1997). In peach, the initial part of the sequence
comprises long distance paradormancy (influence of tissues
situated > 5 cm from the meristematic zone), followed by short
distance paradormancy (influence of tissues situated between
0 and 5 cm from the meristematic zone) and then
endodormancy (Balandier et al. 1993, Rageau et al. 1995).
Later, short distance paradormancy followed by long distance
paradormancy could occur before strict control by ecodormancy.
The involvement of energy metabolism and especially purine metabolism in the endodormancy of buds is well documented (Gendraud 1977, Le Floc’h and Lafleuriel 1981, Le
Floc’h 1984, Gendraud and Petel 1990, Dennis 1994, Le
Floc’h and Faye 1995, Lecomte et al. 1998). This metabolism
produces AMP that is either converted to ATP, which is involved in many metabolic processes and energization of membranes, or to non-adenylic triphosphates (NTP), which are
used in the synthesis of molecules needed for growth. This
second pathway is inactive in dormant tissues (Gendraud
1977, Dennis 1994). A test of endodormancy has been developed, the nucleotides test, that compares the rates of synthesis
of ATP and NTP in bud primordia in the presence and absence
of exogenous adenosine (Gendraud 1975). A significant increase in NTP pools obtained after incubation with adenosine
solution compared with water samples indicates that the
primordia possess synthetic activity and are not endodormant.
This test has been applied to the study of vegetative bud dormancy of various species of perennials, including woody species (oak: Barnola et al. 1986, ash: Lavarenne et al. 1982,
peach: Balandier 1992, Balandier et al. 1993), but has proved
unreliable when applied to floral primordia (Bonhomme 1998,
Bonhomme et al. 1999). Also, the test has several limitations.
First, the buds have to be incubated for a relatively long time
(16 h) at a temperature of 10 °C. Therefore, the observed data
may not correspond strictly to the initial state of the primordia
at the time the incubation began, because this state may have
changed during the incubation. Second, to facilitate comparisons, quantities of nucleotides measured are expressed on the
basis of the amount of protein or DNA in the same tissues.
This requires additional assays, making the procedure timeconsuming, costly and error prone. It would therefore be useful to have a method applicable to both vegetative and floral
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BONHOMME, RAGEAU AND GENDRAUD
Materials and methods
the bioluminescence of 10 µl of extract and that of the same
volume of extract after incubation with pyruvate kinase
(Sigma) and PEP in excess (Pradet 1967). The NTP content
was measured as the difference between the ATP content in
10 µl of extract and that obtained after incubation with nucleoside 5′ diphosphate kinase (Boehringer Mannheim, Roche Diagnostics Corp.) and ADP in excess (Sigma). In this case, S/5
buffer containing 0.0125 mM ADP was used. Each measurement was repeated three times. Proteins were assayed by the
method of Bradford (1976) with bovine serum albumin (BSA)
(Sigma) as reference.
Plant material
Calculations and statistical analyses
For the experiment performed under natural conditions during
winters 1994–1995 and 1995–1996, we used 15 peach (variety Redhaven) trees growing in the INRA orchard at
Clermont-Ferrand, France (45° N, 3° E). The trees had been
grafted on GF305 and were 4 years old in 1994. For the cold
deprivation experiment, we used 15 peach trees similar to
those used in the study carried out under natural conditions except that the trees were planted in 200-dm 3 containers filled
with a local mixed peat soil (Limagne). On October 13, 1994,
the trees were transferred from outside to a heated greenhouse
providing temperatures that fluctuated during the day between
15 and 20 °C and remained at 15 °C during the night. The solar
irradiance was slightly lower in the greenhouse than outdoors,
but the photoperiod was similar.
The mean date of bud burst or bloom was taken as the date
when 50% of buds had reached or passed the critical stage
(green young leaves visible out of scales for vegetative buds,
full open petals for floral buds). Phenological stage was assessed every third day for each bud of 50 twigs (five randomly
chosen twigs on 10 of the 15 trees growing under natural conditions).
Nucleotide concentrations were expressed as pmol nucleotides per µg protein of the primordia. Means and standard error
were calculated from replicate assays (between 4 and 10).
Mean values were compared with the Kruskal-Wallis nonparametric test (Sprent 1992) at a significance threshold of
5%.
Nucleotide and protein assays
Results
Buds were sampled monthly (12 vegetative and 12 floral per
treatment) from the mid-section of twigs taken at random from
early October to the time of bud burst. Buds were immediately
frozen in liquid nitrogen, freeze-dried, and kept at –80 °C until
assayed. The lyophilized vegetative and floral buds were dissected, with the aid of a binocular microscope, into meristematic parts with leaf primordia and floral primordia. Scales
were discarded. The meristematic tissues were immediately
weighed and then processed individually, or bulked to provide
1–2 mg of dry matter. The tissue was ground on ice in 80 to
140 µl of 0.6 N perchloric acid (according to the size of the
primordia) by the method of Keppler et al. (1970) as modified
by Gendraud (1975). The ATP was assayed by bioluminescence (Lumat LB 9501, Kit Boehringer ATP CLS II,
Roche Diagnostics Corp., Indianapolis, IN) at pH 7.5 in 10 µl
of diluted initial extract placed in S/5 buffer with 0.15 mM
phosphoenolpyruvate (PEP) (Sigma, St. Louis, MO). The S/5
buffer was prepared from S buffer (0.2 M TRIS[hydroxymethyl]aminomethane, 7.5 mM MgSO4, 125 mM K2SO4,
2.75 mM ethylenediaminetetraacetic acid (EDTA)), diluted
5:1 with ultrapure H2O and adjusted to pH 7.5 with H2SO4. A
standard was obtained by adding 10 pmol of exogenous ATP
(Sigma) to the extract. A zero control was obtained by consuming the ATP present in the extract by incubation in the
presence of hexokinase (Boehringer Mannheim, Roche Diagnostics Corp.) and 180 mg ml –1 glucose to allow for any
quenching effect caused by impurities.
The ADP content was measured as the difference between
Meteorological and phenological characterization of the 2
years of observations
primordia that avoids or minimizes these disadvantages. We
tested the hypothesis that there is a close correlation between
the state of endodormancy of vegetative and floral primordia
and (i) their ATP and NTP concentrations, which could instantaneously characterize the activity of their energy metabolism,
and (ii) the ATP/ADP ratio, which could be considered a
marker of the degree of oxidative phosphorylation of the tissues.
In autumn and winter 1994–1995, the temperature fell
steadily until mid-December. A cold period persisted from
mid-December to mid-January (Figure 1). From mid-January
onward, daily mean temperatures exceeded 5 °C and increased
until bud break. The mean dates of burst of terminal and
axillary vegetative buds were February 10 and 17, respectively. For floral buds, the mean date of bloom was March 20.
In 1995–1996, the first cold period lasted from the middle
of November until the end of December. After a warmer spell
in January, another cold period extended from the end of Janu-
Figure 1. Ten-day mean temperatures for fall and winter 1994–95 and
1995–96.
TREE PHYSIOLOGY VOLUME 20, 2000
ATP/ADP RATIO AND THE DORMANCY STATE OF PEACH BUDS
ary to March 13. The mean dates of bud burst of terminal and
axillary vegetative buds were March 18 and 24, respectively.
For floral buds, the mean date of bloom was March 30. Based
on the dynamic model of Fishman et al. (1987a, 1987b) with
September 1 as the starting date, we estimated that endodormancy ended on December 15, 1994 and December 20, 1995.
Dynamics of nucleotide concentrations and ATP/ADP ratio
under natural conditions
In 1994–1995, the ATP concentration in vegetative primordia
was about 4 pmol µg –1 protein at the beginning of October
(Figure 2a). The ATP concentration then decreased slowly until mid-January (30% in 3 months). This decline was then followed by a rapid increase in ATP concentration (threefold in
1 month). In 1995–1996 (Figure 2b), at the beginning of October, the ATP concentration was similar to that observed in
1994–1995. A small net increase occurred in mid-December.
After a transitory decrease in early January, the increase resumed until mid-March.
In 1994–1995, the ADP concentration in vegetative primordia was equivalent to the ATP concentration until mid-January. However, unlike the ATP concentration, it increased only
slightly and did not exceed the concentration observed at the
beginning of October. In mid-February, shortly before bud
break, the ADP concentration was less than half the ATP con-
617
centration. In 1995–1996, the ADP concentration was equivalent to, or slightly less than, the ATP concentration from early
October to early January. It more or less tracked the changes in
ATP concentration until March, when ATP concentration increased, whereas ADP concentration remained unchanged.
Consequently, in mid-March, just before bud break, as in
1995, the ADP concentration was about half that of the ATP
concentration.
In 1994–1995, the ATP concentration in floral primordia
was about 1 pmol µg –1 protein at the beginning of October
(Figure 2c). An increase in ATP concentration began in
mid-December. The increase began gradually and then became rapid and sharp in February (3.5-fold increase in
15 days). In 1995–1996 (Figure 2d), in early October, the ATP
concentration was approximately twice that observed in
1994–1995. An increase in ATP concentration started at the
beginning of January and continued until mid-March.
In floral primordia, in 1994–1995, the ADP concentration
decreased from the beginning of October to the end of January. At the end of January, the ADP concentration increased
rapidly (3.2-fold in 15 days). From October until mid-January,
the ADP concentration was higher than the ATP concentration, at first appreciably (October and November), then only
slightly (December and beginning of January); however, by
the end of January the ADP concentration was less than the
Figure 2. Dynamics of adenylic diphosphate (ADP), adenylic triphosphate
(ATP) and non-adenylic triphosphate
(NTP) nucleotide concentrations, expressed on a protein basis, in vegetative
(a and b) and floral (c and d) primordia
of peach buds on trees growing under
natural conditions during winters
1994–1995 (a and c) and 1995–1996
(b and d). Bars represent standard errors.
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BONHOMME, RAGEAU AND GENDRAUD
ATP concentration. In mid-February, the ADP concentration
was approximately 60% of the ATP concentration. In
1995–1996, the ADP concentration was higher than the ATP
concentration from the beginning of October to mid-December. From mid-December to mid-February it was stable and
lower than the ATP concentration. It then increased rapidly at
the beginning of March but remained lower than the ATP concentration.
In both 1994–1995 and 1995–1996, NTP concentrations in
vegetative and floral primordia changed in proportion to ATP,
but were between 30 and 50% lower than the ATP concentrations. In floral primordia, minimum NTP concentrations were
about 25% of those measured in vegetative primordia
(0.5 pmol µg –1 protein in 1994–1995, 1 pmol µg –1 in 1995–
1996).
In vegetative primordia, the ATP/ADP ratio remained constant from early October until mid-January in 1994–1995 and
until the end of January in 1995–1996 (Figure 3a) with values
close to 1 in 1994–1995 and 1.4 in 1995–1996. Thereafter, the
ratio increased rapidly, approaching 2. In floral primordia
(Figure 3b), the ratio was initially low (< 0.5) and then increased sharply at the end of January in 1995, and slightly earlier (end of December) in 1996. It approached 1.5 and then
stabilized.
Dynamics of nucleotide concentrations and ATP/ADP ratio
during cold deprivation
In both vegetative (Figure 4a) and floral (Figure 4b)
primordia, the concentrations of ATP, ADP, and NTP slowly
decreased throughout the cold deprivation treatment. The decrease in ADP concentration in vegetative primordia began
only in late November, after the ATP and NTP concentrations
had already decreased. Unlike the buds of the trees grown under natural conditions, no increases in nucleotide concentrations occurred in buds of trees in the cold deprivation
treatment at the end of the sampling period. Necrosis of the
floral primordia began soon after the last date of sampling.
In vegetative primordia in the cold deprivation treatment,
the ATP/ADP ratio decreased between October 4 and November 15, and then stabilized at this low value (Figure 5a). In floral primordia in the cold deprivation treatment, the ATP/ADP
ratio remained at the low initial value (Figure 5b).
Discussion
We observed changes in nucleotide concentrations in vegetative and floral buds from January onward, and consequently
changes in the values of the ATP/ADP ratio. The rate at which
this ratio changed seemed to depend on the year, but similar
peak values were reached shortly before bud break in both
years of study.
Vegetative primordia in natural conditions
In vegetative buds of strawberry, Robert (1996) observed a
strong and rapid increase in ATP concentration that was correlated with the end of endodormancy, based on the nucleotides
test. In contrast, we could not correlate the end of endodormancy with either the ATP concentration or the dynamics
Figure 3. Dynamics of ATP/ADP ratio in
vegetative (a) and floral (b) primordia of
peach buds on trees growing under natural
conditions during winters 1994–1995 and
1995–1996. Bars represent standard errors.
Figure 4. Dynamics of adenylic diphosphate (ADP), adenylic triphosphate (ATP)
and non-adenylic triphosphate (NTP) nucleotide concentrations, expressed on a
protein basis, in vegetative (a) and floral
(b) primordia of cold-deprived peach buds
during winter 1994–1995. Bars represent
standard errors.
TREE PHYSIOLOGY VOLUME 20, 2000
ATP/ADP RATIO AND THE DORMANCY STATE OF PEACH BUDS
619
Figure 5. Dynamics of ATP/ADP ratio
in vegetative (a) and floral (b) primordia
of peach buds on trees grown under natural conditions or subjected to cold deprivation during winter 1994–1995. Bars
represent standard errors.
of the ATP/ADP ratio in vegetative and floral buds of peach
trees. The results of the nucleotides test indicated that endodormancy ended before the end of December (authors’ unpublished data), whereas changes in ATP concentration and
ATP/ADP ratio did not occur until after the end of January
(Figures 2 and 3).
On the other hand, the dynamics of the changes shown in
Figures 2 and 3 were consistent with results obtained from the
one-node cutting test applied on buds of Redhaven trees cultivated under natural conditions over several years at Clermont-Ferrand (Rageau et al. 1995). These results showed that
endodormancy and short distance paradormancy end no earlier than December 20 and no later than January 15, and generally within the first 10 days of January (Balandier et al. 1993,
Rageau et al. 1995). Endodormancy and short distance paradormancy could not be differentiated by the one-node cutting
test (Rageau et al. 1995). We found that the increase in the
ATP/ADP ratio began very shortly after the end of endodormancy and short distance paradormancy. The time needed
for the ATP/ADP ratio to increase was dependent on the temperature following the onset of the increase (mean temperature
was about 8 °C from January 15 through February 20, 1995;
mean temperature was below 4°C from January 28 through
March 8, 1996; Figure 3a). However, it is likely that once initiated, the changes in nucleotide concentrations and ATP/ADP
ratio are irreversible. Therefore, the increase in ATP/ADP ratio could indicate a major change in the physiological state of
the buds. We conclude that the change in ratio is an indication
of bud growth potential rather than an indication of the actual
growth rate, which depends on the prevailing temperature.
The ATP/ADP ratio did not change before January, indicating that it is not correlated with the progressive accumulation
of chilling. This means that the deep changes in the bud state
take place rapidly, but only after the trees have received a critical amount of chilling. This amount corresponds not only to
endodormancy release, which should occur in late December
as estimated by the dynamic model of Fishman et al. (1987a,
1987b) (December 15, 1994 and December 20, 1995, respectively), but also to short distance paradormancy release, which
requires additional chilling. Because the changes in ATP/ADP
ratio occurred later than strict endodormancy release, they
provide a marker for the end of both endodormancy and short
distance paradormancy.
For practical use, we propose a threshold value of 1.5 for the
ATP/ADP ratio, below which vegetative primordia are in a
state of endodormancy or paradormancy, or both. This threshold is easier to locate on the curve than the beginning of the increase in ATP/ADP ratio (Figure 3).
Floral primordia in natural conditions
Until mid-December, the ATP concentration remained low
relative to the ADP concentration. This is a major difference
between floral and vegetative primordia. On the other hand, as
in vegetative primordia, the NTP concentration remained between 30–50% of ATP concentration until early January.
Early January is the time when peach floral primordia usually
recover their full growth capacity (Rageau 1982). Thus, the
shift in ATP/ADP ratio is consistent with the time when only
ecodormancy controls floral bud growth. This time can be verified by forcing twigs at 25 °C over a 7-day period (Tabuenca
1964).
In 1996, changes in the ATP/ADP ratio in floral buds started
to increase earlier than in vegetative buds. We observed a similar rate of increase in the ATP/ADP ratio in both 1995 and
1996, possibly because the temperature pattern following the
onset of the ATP/ADP ratio increase in floral buds was similar
in both years. In contrast, the temperature pattern following
the onset of the increase in ATP/ADP ratio in vegetative buds
differed between the two years.
The ATP/ADP ratio of floral buds remained close to 0.5 and
was always less than 1.0 during the period between early October and late December, whereas it was close to 1.5, and always greater than 1.0, after the resumption of growth capacity
in early January. A threshold value of 1.0 was therefore selected as a marker of this change in the state of floral buds.
The low ATP and NTP concentrations in floral buds between early October and late December corresponded to an
unfavorable metabolic state impeding growth, which could
partly explain why the nucleotides test cannot be applied to
floral buds (Bonhomme 1998, Bonhomme et al. 1999). We
hypothesize that, during the nucleotides test, the supply of
adenosine preferentially increases the ADP concentration.
The lack of ATP (or its slow regeneration) may result in insufficient energy to carry out phosphorylation of enough
non-adenylic diphosphate molecules to allow an increase in
NTP concentration. The immediate utilization of the ATP synthesized to provide the energy needed for the different stages
of the biosynthesis of NTP would also explain the absence of
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BONHOMME, RAGEAU AND GENDRAUD
an increase in ATP concentrations in the primordia incubated
with adenosine during the test.
The increase in ATP/ADP ratio in both vegetative and floral
buds corresponded fairly well with the transition from a state
of endo- or paradormancy to a state free of either dormancy.
Only the rate of this change depends on the prevailing temperature. Therefore, the value of the ATP/ADP ratio could allow
discrimination between endodormancy or paradormancy, or
both, and ecodormancy.
Vegetative and floral primordia during cold deprivation
Results of the one-node cutting test on Redhaven peach trees
subjected to cold deprivation indicated that vegetative buds remained under endodormancy or short distance paradormancy,
or both, well beyond February (mean time for bud break was
about 20 days, which is above the mean value of 12 days for
Redhaven peach buds under natural conditions) (authors’ unpublished data). The stability of the ATP/ADP ratio below the
threshold value of 1.5 is consistent with the persistence of
endodormancy or short distance paradormancy, or both, in
vegetative buds subjected to cold deprivation.
Based on the dynamics of fresh and dry weights of floral
primordia obtained over several years under natural conditions
and during cold deprivation (Bonhomme 1998), we conclude
that floral primordia are unable to grow while the cold deprivation treatment is applied. The cold deprivation treatment
caused the death of the primordia after about 6 months. The
prolonged period of growth incapacity was paralleled by a
prolonged period when the ATP/ADP ratio remained below
the critical threshold of 1.0. The dynamics of the nucleotide
concentrations indicated that the initial energy status of the
primordia was unfavorable for growth, and showed a tendency
to worsen in response to cold deprivation. Thus, the floral
primordia contained lower ATP concentrations and ATP/ADP
ratios than the vegetative buds, which may partly explain why
vegetative primordia can survive cold-deprived conditions
whereas floral buds cannot.
We conclude that the ATP/ADP ratio in buds of woody
plants cannot be used as a marker of endodormancy. However,
the ratio can be used to characterize buds in endodormancy
and paradormancy, and it is a good marker of their growth capacity in situ. Because the test is rapid and simple to implement, and does not require that the buds be incubated, it is
applicable to both floral and vegetative buds. Tests are now in
progress to determine if a decrease in ATP/ADP ratio is correlated with induction of dormancy in late summer.
Acknowledgments
We thank J.P. Richard and N. Jallut for assistance and ATT for checking the English.
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