Annals of Botany 91: 429±438, 2003
doi:10.1093/aob/mcg037, available online at www.aob.oupjournals.org
Time-course of Tomato Whole-plant Respiration and Fruit and Stem Growth
During Prolonged Darkness in Relation to Carbohydrate Reserves
C . G A R Y 1 , * , P. B A L D E T 2 , N . B ER T IN 1 , C . D E V A U X 2 , M. T C H A M I T C H I A N 1 and P . R A Y M O N D 2
1INRA, Unite
 Plantes et SysteÁmes de culture Horticoles, Domaine St Paul, Site Agroparc, 84914 Avignon Cedex 9,
France and 2INRA, UMR Physiologie et Biotechnologie VeÂgeÂtales, IBVM, Centre de Recherches de Bordeaux
Aquitaine, 71 avenue E. Bourleaux, BP 81, 33883 Villenave d'Ornon Cedex, France
Received: 19 June 2002 Returned for revision: 1 October 2002 Accepted: 16 November 2002 Published electronically: 23 January 2003
To evaluate the relevance of a simple carbon balance model (Seginer et al., 1994, Scientia Horticulturae 60:
55±80) in source-limiting conditions, the dynamics of growth, respiration and carbohydrate reserves of tomato
plants were observed in prolonged darkness. Four days prior to the experiments, plants were exposed to high or
low light levels and CO2 concentrations. The concentration of carbohydrates in vegetative organs was 30±50 %
lower in plants that were exposed to low carbon assimilation conditions compared with those exposed to high
carbon assimilation conditions. During prolonged darkness, plants with low carbohydrate reserves exhibited a
lower whole-plant respiration rate, which decreased rapidly to almost zero after 24 h, and carbohydrate pools
were almost exhausted in leaves, roots and ¯owers. In plants with high carbohydrate reserves, the whole-plant
respiration rate was maintained for a longer period and carbohydrates remained available for at least 48 h in
leaves and ¯owers. In contrast, fruits maintained fairly stable and identical concentrations of carbohydrates and
the reduction in their rate of expansion was moderate irrespective of the pre-treatment carbon assimilation conditions. The time-course of asparagine and glutamine concentrations showed the occurrence of carbon stress in
leaves and ¯owers. Estimation of source and sink activities indicated that even after low carbon assimilation,
vegetative organs contained enough carbohydrates to support fruit growth provided their own growth stopped.
The time of exhaustion of these carbohydrates corresponded grossly to the maintenance stage simulated by the
model proposed by Seginer et al. (1994), thus validating the use of such a model for optimizing plant growth.
ã 2003 Annals of Botany Company
Key words: Tomato, Lycopersicon esculentum Mill., prolonged darkness, respiration, carbohydrate pools, fruit growth,
carbon stress, source±sink balance.
INTRODUCTION
The growth of plants, i.e. the production of new structures,
results from the acquisition of resources (mainly carbon),
their temporary storage and/or translocation to the growing
tissues. The activities of source or sink organs are regulated
in different ways so that crop growth and yield are either
source-limited or sink-limited (Ho et al., 1989). The status
of carbohydrate pools may be considered to be an indicator
of the source±sink balance. In conditions where sources are
limiting, the size of carbohydrate pools is reduced and,
when there is a shortage of carbon substrate, rates of
biosyntheses decrease. In conditions where sinks are
limiting, carbohydrates are stored and can inhibit the
production of new assimilates (AzcoÂn-Bieto, 1983).
Seginer et al. (1994) formalized these concepts in a
simple carbon balance model with two state variables (the
masses of carbon in a single carbohydrate pool and in plant
structures) and four carbon ¯uxes (gross photosynthesis,
maintenance respiration, growth respiration and production
of structural biomass). The carbohydrate pool acts as a
buffer with a limited capacity. When it is reduced in size,
* For correspondence at: UMR System (Agro.M-CIRAD-INRA),
CIRAD, Campus Lavalette, TA 40/01, Avenue Agropolis, 34398
Montpellier Cedex 5, France. Fax +33 (0)4 67 61 55 12, e-mail
[email protected]
growth continues as long as gross photosynthesis is higher
than maintenance respiration. If this condition is not met,
structural compounds are mobilized to maintain a minimum
value of respiration which, in the model, is equivalent to a
negative growth rate. When the carbohydrate pool reaches
its highest value, the photosynthetic in¯ow is limited to the
out¯ow of carbohydrates consumed by respiration and
growth. In this framework, optimizing climate control in
glasshouses to maximize plant growth is possible. To this
end, the carbohydrate pool should remain within its bounds;
if it is not then growth is limited either by source or sink
activity (Tchamitchian and Ioslovich, 2000).
In a glasshouse, climate is modi®ed in such a way that the
source±sink balance is altered (Gary and Baille, 1999). The
glass limits the availability of light and the replenishment of
CO2 assimilated by the crops (except when CO2 enrichment
is possible) and, therefore, source activity. The increased
temperature that results from the reduction in radiative and
convective heat loss and, possibly, from heating of the
glasshouse increases the potential growth rate and therefore
the sink activity. De Koning (1994) estimated that the
actual growth rate of glasshouse tomato plants is equal, on
average, to about half the sink strength of their vegetative
and reproductive organs. Under these conditions, glasshouse-grown crops should be more prone to a shortage,
ã 2003 Annals of Botany Company
430
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
rather than excess, of carbohydrates. Therefore, the present
study focuses on the dynamics of respiration rate, carbohydrate pools and organ growth, in the absence of
photosynthetic activity, in plants with contrasting amounts
of initial carbohydrate reserves.
In prolonged darkness, plant tissues go through a
sequence of stages (Baysdorfer et al., 1988; Brouquisse
et al., 1991). First, the carbohydrate concentration and rates
of respiration and growth decrease. Non-carbohydrate
compounds are then used as substrates for the declining
respiratory activity. Finally, irreversible processes such as
the degradation of membranes occur, leading to cell death.
During the ®rst stage, if the decrease in carbohydrate
content and respiration rate were concomitant, then the
respiration rate would actually be limited more by the rates
of biosynthesis and consequent demand for ATP
(Brouquisse et al., 1991) than by carbohydrate availability.
A simultaneous decrease in growth and respiration rates has
been observed, e.g. in pea (Hole and Scott, 1984) and
tomato (Grange and Andrews, 1995) fruits, and in alfalfa
(Hendershot and Volenec, 1989) and tall fescue (Moser
et al., 1982) leaves. In wheat and spinach leaves, dark
respiration is correlated to the previous net assimilation of
CO2 and to carbohydrate concentration (AzcoÂn-Bieto and
Osmond, 1983; AzcoÂn-Bieto et al., 1983). In wheat, the
duration of dark treatment before the leaf extension rate
(LER) starts to decrease depends on the light intensity of the
previous day (Christ, 1978a). Sambo (1983) showed that in
two grass species there is a critical assimilate level below
which leaf growth is limited.
During the second stage of carbon starvation, substrates
other than carbohydrates can be metabolized. This consumption of structural compounds can be deduced from the
time-course of the carbon balance of whole vegetative
tomato plants: at temperatures of 20 and 25 °C and after 24 h
darkness, carbon losses by respiration were larger than the
decrease in carbon content in the carbohydrate pools (Gary,
1989). At the cell scale, proteolysis was observed in maize
plantlets (Brouquisse et al., 1998), and beta-oxidation of
fatty acids in root tips (Dieuaide et al., 1993). Cells develop
a survival strategy involving controlled autophagy. In
sycamore cells growing in a sucrose-free medium, the
number of mitochondria per cell decreased (Journet et al.,
1986). It was hypothesized that the products of the
breakdown of some mitochondria supplied respiratory
substrates for the remaining mitochondria. Ultimately, in
the third stage, this process becomes irreversible and leads
to cell death.
Prolonged darkness has been used by crop modellers to
estimate the maintenance component of respiration
(Amthor, 1989). This `dark decay method' is based on the
hypothesis that, after a few hours of darkness, growth (i.e.
the production of new structures) stops and the remaining
respiratory activity supports only the energy cost of
functions such as protein turnover and maintenance of
ionic gradients. This hypothesis has been veri®ed only for
the transition between the ®rst and second stages of carbon
starvation (Gary, 1989). Later, metabolism contributes to
the survival and no more to the maintenance of cell
structures. Therefore, this transient stage is the time when
the plant carbohydrate pool reaches its minimum value in
the model of Seginer et al. (1994). To date, this model has
been calibrated by using experimental data from young
vegetative tomato plants only. Hence, an experiment was
designed to assess the behaviour of the carbon balance of
fruiting tomato plants. Relationships between the dynamics
of respiration rate, carbohydrate pools and growth rate of
organs in prolonged darkness were studied to identify the
conditions under which carbon stress occurred at the wholeplant level and in vegetative and reproductive organs.
Indicators of carbon stress, such as accumulation of the
amino acid asparagine (Brouquisse et al. 1992; Chevalier
et al., 1996), were examined.
MATERIALS AND METHODS
Cultivation of plants
Tomato seeds (Lycopersicon esculentum Mill. `RaõÈssa')
were sown in small rockwool cylinders on 4 Mar. 1997
(expt 1) and 21 Apr. 1997 (expt 2). Once three leaves had
appeared, seedlings were transplanted to a multispan
glasshouse located in southern France (INRA, Avignon).
Plants were grown in a recirculating hydroponic system
under the same conditions as described by Gary et al.
(1998). All axillary buds were removed, and six fruits were
retained per truss. When the ®fth truss ¯owered, plants were
transplanted to two similar growth chambers (8´75 m2) for
10±12 d periods starting on 22 May (expt 1) and 3 July
(expt 2). In both experiments, plants in one growth chamber
were used for gas exchange measurements, and those in the
other for growth measurements and tissue analyses. In each
growth chamber, 12 plants were set in a recirculating
hydroponic system consisting of 12 3 10-l pots linked to a
300-l reservoir in which the nutrient solution was circulated
continuously. The nutrient solution contained the following
major elements: 4´0 mol m±3 KNO3, 0´9 mol m±3 K2SO4,
1´5 mol m±3 KH2PO4, 3´75 mol m±3 Ca(NO3)2 and 1´0 mol
m±3 MgSO4. pH was checked daily and varied between 5´5
and 6´5. Air temperature, CO2 concentration and humidity
were controlled and recorded; metal halide lamps were used
to provide arti®cial lighting.
Climatic treatments
The experiments were designed to load plant carbohydrate reserves to a high (expt 1) or a low (expt 2) level
before subjecting the plants to a long period of darkness. For
this purpose, radiation values were set so that their daily
integral was equal to mean values measured in Avignon,
France (43°55¢N, 4°52¢E) in July and January, respectively,
and CO2 concentration was increased in expt 1.
Experiment 1. For 4 d, arti®cial lighting was provided
between 0500 and 2100 h [1000 mmol m±2 s±1 PAR
(photosynthetically active radiation) for the ®rst 3 d and
500 mmol m±2 s±1 PAR for the fourth day]; air temperature
was set to 20 °C in the light and 16 °C in the dark. The CO2
concentration was controlled at approx. 960 ppm and
humidity was maintained between 70 and 80 %. At the end
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
of the fourth day, lights were turned off for 4 d, and the
temperature was set to 20 °C; the CO2 concentration varied
between 350 and 600 ppm owing to plant respiration and
ventilation.
Experiment 2. As for expt 1, except that PAR was set to
200 mmol m±2 s±1 and CO2 concentration to 350 ppm during
the ®rst 4 d.
Measurement of CO2 exchange
One of the growth chambers was used as a closed system
in which to measure CO2 exchange of a set of 12 plants.
During daylight, CO2 was injected into both growth
chambers to compensate for photosynthesis and maintain
the set-point value of CO2 concentration. In the period of
darkness, the CO2 concentration increased as a result of
plant respiration; when [CO2] reached 600 ppm the growth
chambers were ventilated using air from outside the
building to reduce the [CO2] to approx. 350 ppm. As the
growth chambers were not perfectly airtight, a leakage
coef®cient (K, m3 s±1) was estimated for both chambers
prior to each experiment. For this purpose, CO2 was injected
into an empty and closed growth chamber up to 1000 ppm,
and the dynamics of the decrease in CO2 concentration, Ci(t)
(in ppm), was analysed. The CO2 balance of the growth
chamber was:
V
dCi …t†
ˆ K…Co ÿ Ci …t††
dt
…1†
where V (m3) is the volume of the growth chamber and Co
(ppm) is the CO2 concentration outside the growth chamber.
Then:
Kt
Ci …t† ˆ Co ‡ …Ci …t† ÿ Co †e ÿ V
…2†
The leakage coef®cient, K, was calculated by non-linear
regression. During the experiment, at any time except when
a growth chamber was ventilated, its CO2 balance was:
V
dCi …t†
ˆ N ‡ I ‡ K…Co ÿ Ci …t††
dt
…3†
where N (m3 s±1) is the CO2 exchange rate of the plant
and I (m3 s±1) is the CO2 injection rate. N can be
calculated as:
N ˆ ÿI ÿ K…Co ÿ Ci …t ÿ Dt†† ÿ
K…Ci …t† ÿ Ci …t ÿ Dt††
ÿK
1 ÿ e V Dt
…4†
where Dt (300 s in both experiments) is the time step of CO2
measurements (Gary, 1988a).
431
Measurement of fruit and stem growth
Variations in fruit diameter were recorded simultaneously
on six fruits during their period of maximum growth rate,
i.e. 2 to 4 weeks after ¯ower anthesis (fruit diameter around
50 mm). Variations in stem diameter were recorded
simultaneously on six plants, on internodes exhibiting
their maximum growth rate, i.e. internodes located beneath
the ¯owering truss. Measurements were made using lineardisplacement transducers (CD 4112-1; Schlumberger
Enertec, VeÂlizy-Villacoublay, France) with a precision of
63 mm.
Carbohydrate and amino acid analyses
Every 12 h during the period of prolonged darkness,
samples of young leaves, ¯owers, roots and fruits from two
plants were harvested for analysis. Lea¯ets were randomly
sampled among the terminal and top-pair lea¯ets on one leaf
of the fourth or ®fth sympod, i.e. close to the last ¯owering
cluster. The ®ve or six available ¯owers per plant were
pooled. Root tissue (approx. 10 g f. wt) was rinsed in
distilled water. Since the timing of ¯owering had been
noted, the age of each fruit was known. On each plant, fruits
that were 4±5 and 14±16 DAA (days after anthesis) old were
harvested for analyses. All samples were quickly frozen in
liquid nitrogen and stored at ±80 °C until analysis. Dry
weight was determined after freeze-drying. Soluble carbohydrates were extracted and starch hydrolysed according to
Brouquisse et al. (1991), and enzymatic analyses were
carried out using the method of Kunst et al. (1984). Free
amino acids were analysed by HPLC according to Cohen
and De Antonis (1994).
RESULTS
Status of plants submitted to 4 d of high and low carbon
assimilation
During the 4 d pre-treatment, the quite different conditions
of light and CO2 concentration led to contrasting rates of
CO2 exchange, but rates of increase in fruit diameter were
more similar (Table 1). Compared with that in plants
subjected to high carbon assimilation conditions, net
photosynthesis was reduced by 7´9-fold and night respiration by 6´5-fold in plants pre-treated in low carbon
assimilation conditions. Measurements of net photosynthesis in the day and respiration at night were consistent
with values predicted by the model of Seginer et al. (1994).
When using the set of parameters adopted by these authors
and the mean plant dry weight and leaf area index observed
in expts 1 and 2, calculated net photosynthesis was 10´3 and
2´4 mmol per plant s±1 in high (1000 mmol m±2 s±1 PAR,
960 ppm CO2) and low (200 mmol m±2 s±1 PAR, 300 ppm
CO2) carbon assimilation conditions, respectively.
Calculated night-time respiration was 2´4 and 0´5 mmol
per plant s±1 under conditions of high and low carbon
assimilation, respectively.
As expected, the status of the carbohydrate pools in the
vegetative and reproductive organs differed between both
pre-treatments (Table 2). In the vegetative organs (roots and
432
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
growing leaves), concentrations of soluble carbohydrates
and starch were 30±50 % lower following exposure to low
rather than high carbon assimilation conditions. In contrast,
in ¯ower buds and growing fruits, there were no signi®cant
differences in soluble carbohydrate concentration whereas
the starch pool dropped by 50±84 % in plants pre-treated in
high compared with low carbon assimilation conditions.
Despite these quite different net assimilation rates and
carbohydrate reserves, the expansion rate of fruits from both
treatments did not differ dramatically (Table 1). On average,
it tended to be higher during the day and lower at night (but
not signi®cantly so) under conditions of low compared with
high carbon assimilation.
Time-course of whole-plant respiration rate during
prolonged darkness
During the ®rst 24 h of darkness, the average respiration
rate was similar to that of the four previous nights: 1´58 and
0´21 mmol per plant s±1 following high and low carbon
assimilation pre-treatments, respectively (Fig. 1). The
TA B L E 1. CO2 exchanges and organ growth rates during
the pre-treatment period
High C
assimilation
Daytime
Net photosynthesis (mmol per
plant s±1)
Fruit growth (mm h±1)
Stem growth (mm h±1)
11´3
Night-time
Respiration (mmol per plant s±1)
Fruit growth (mm h±1)
Stem growth (mm h±1)
Low C
assimilation
1´4
29´6 (8´7)
2´6 (1´9)
40´1 (9´4) n.s.
±
1´2
36´2 (8´7)
11´4 (3´8)
0´2
27´8 (7.0) n.s.
±
All variables were averaged over the three day and night periods prior
to the period of prolonged darkness. In the high radiation treatment,
photosynthesis rate was averaged over the three daytime periods at
1000 mmol m±2 s±1 PAR. Measurements of stem diameter were not
available in the low radiation pre-treatment.
CO2 exchanges, mean of 12 plants. Organ growth rates, mean (s.d.) of
measurements from six sensors.
n..s, Non signi®cant (P > 0´05).
average respiration rate remained high in plants subjected
to the high carbon assimilation pre-treatment, whereas it
dropped by 60 % during the ®rst 6 h then levelled off in
plants pre-treated in low carbon assimilation conditions.
After approx. 16 h of darkness, the respiration rate
decreased exhibiting a periodic pattern of a sharp decrease
followed by a plateau and/or a slight increase. After high
carbon assimilation, this pattern was observed three times,
starting after approx. 16, 40 and 67 h of darkness. In this
experiment (expt 1), the last plateau levelled off after 79 h of
darkness at 0´19 mmol per plant s±1, i.e. at about 12 % of the
initial value and at about the same value as that observed
initially in plants that had experienced conditions of low
carbon assimilation (expt 2). In the latter plants, the
respiration rate dropped after 16 h darkness, reaching very
low values (close to the threshold of sensitivity of the CO2
exchange measurement system) after 24 h.
Time-course of carbohydrate and amino acid concentrations
in leaves, roots and growing fruits during prolonged
darkness
In tomato leaves, starch was the major carbohydrate pool.
After high carbon assimilation, its concentration decreased
steadily until it was depleted after 48±60 h of darkness
(Fig. 2). Meanwhile, the concentration of soluble carbohydrates decreased slowly, remaining above 60 mmol g±1 d. wt
after 84 h of darkness. Following low carbon assimilation,
the leaf starch pool was depleted within 12 h of darkness and
the soluble carbohydrate concentration dropped and remained below 50 mmol g±1 d. wt after 24 h of darkness.
Tomato roots stored little starch (less than 20 mmol g±1
d.wt). Their concentration of soluble carbohydrates decreased steadily irrespective of the light and CO2 pretreatment, reaching values of less than 50 mmol g±1 d. wt
after 36 h of darkness.
After high carbon assimilation, the starch concentration
of ¯owers decreased by 80 % in 24 h while the concentration of soluble carbohydrates remained above 110 mmol
g±1 d. wt throughout the 84 h of prolonged darkness. After
low carbon assimilation, the starch pool quickly reached an
all-time low level, whereas the concentration of soluble
carbohydrates decreased steadily to below 50 mmol g±1 d. wt
after 48 h of darkness.
TA B L E 2. Concentrations (nmoles glucose equivalents mg±1 d. wt) of soluble carbohydrates and starch at the beginning of
the period of prolonged darkness, following 4 d of high and low carbon assimilation pre-treatments
High C assimilation
Roots
Young leaves
Flower buds
Fruits 5 DAA
Fruits 15 DAA
Low C assimilation
Soluble carbohydrates
Starch
Soluble carbohydrates
Starch
150
195
195
909
2282
20
1117
253
459
841
86
105
194
763
2200
14
563
59
121
423
(19)
(28)
(18)
(132)
(158)
(1)
(433)
(86)
(122)
(168)
Mean (s.d.) of eight analyses: two plants 3 two extractions 3 two analyses.
** P < 0´01; * P < 0´05; n.s., non signi®cant.
(23)**
(6)**
(9) ns
(41)*
(73) ns
(2)**
(51)**
(5)**
(74)**
(152)**
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
In contrast to all other organs, growing fruits exhibited no
changes in carbohydrate concentration throughout the
period of prolonged darkness, irrespective of treatment.
They stored more soluble carbohydrates than starch, with
concentrations ®ve to ten times higher than those in ¯ower
buds or vegetative organs. During the cell division stage, the
concentration of soluble carbohydrates was slightly higher
(mean 1230 vs. 1090 mmol g±1 d. wt) and the concentration
of starch slightly lower (mean 175 vs. 318 mmol g±1 d. wt)
after low than after high carbon assimilation. During the cell
expansion stage, the concentration of soluble carbohydrates
was even higher, decreasing slowly from 2200 to 1700 mmol
g±1 d. wt. The starch concentration remained stable, at
around 840 and 440 mmol g±1 d. wt after high and low
carbon assimilation, respectively.
In leaves, the concentrations of asparagine and glutamine
started increasing after 36 h of darkness, irrespective of
previous carbon accumulation (Fig. 3). Both amino acids
were present at low concentrations in roots throughout the
period of prolonged darkness. In ¯owers, as in leaves, the
asparagine concentration increased signi®cantly after 36 h
of darkness, and the rate of increase following high carbon
assimilation was twice that following low carbon assimilation. In contrast, the glutamine concentration increased only
very gradually throughout the period of darkness. The
amino acid concentration was higher in fruits than in
vegetative organs; concentrations of both asparagine and
glutamine increased slowly and did not exhibit any dramatic
change.
Time-course of the growth rate of fruits and stems during
prolonged darkness
Despite large differences in carbohydrate storage and
respiratory activity between plants in the two treatments, the
growth rate of fruits was only slightly higher (not always
signi®cantly) after high than after low carbon assimilation
(Fig. 4A). The change in fruit growth was comparable with
the change in whole-plant respiration rate: it was high and
¯uctuated widely during the ®rst 16 h (averaging 43´1 and
F I G . 1. Time-course of whole-plant respiration rate during prolonged
darkness and after 4 d of high (open symbols) and low (closed symbols)
carbon assimilation. CO2 ef¯ux was measured every 5 min on a set of 12
plants located in a single growth chamber, and averaged on an hourly
basis.
433
36´6 mm h±1 after high and low carbon assimilation,
respectively), then decreased by 41 % during the next 16 h
and by another 21 % during the following 50 h. At the end of
the experiment, i.e. after 82 h of darkness, the fruit growth
rate was still about one-third of their growth rate during
the day±night cycles, regardless of the light and CO2
concentration conditions.
The changes in stem growth rate were more erratic
(Fig. 4B). Unexpectedly, it tended to be lower during the
®rst 40 h (yet not always signi®cantly so), then higher in
plants with high carbohydrate reserves than in those with
low carbohydrate reserves. It decreased slightly and almost
stopped after 70 (high carbon assimilation plants) or 50 h
(low carbon assimilation plants). However, these observations cannot be considered conclusive since the range of
hourly stem growth rates was close to the sensitivity of the
displacement transducers.
DISCUSSION
The effects of prolonged darkness clearly differed with the
initial size of the carbohydrate pools. Following high carbon
assimilation, the high carbohydrate content could support
growth and respiration for 20±24 h at the high levels
observed during the previous day/night periods; both
processes then decreased. Following low radiation conditions, carbohydrate availability was lower, and the respiration rate was maintained for 20±24 h at the low levels
experienced during the previous day/night periods before a
rapid decline to almost undetectable levels.
Such a response of plant respiration to assimilate
availability has often been described in the literature (see
review by Amthor, 1989). This response can be explained
on two time scales. In the short term, a reduction in the
supply of substrate limits overall metabolism. In the long
term (i.e. a few hours to a few days), the respiratory capacity
gradually acclimates to the new ¯ux of assimilate supply.
Williams et al. (1992) observed changes in the pattern of
protein synthesis in barley roots only 24 h after shoot
pruning; activities of invertase and sucrose synthase had
declined. In similar conditions, McDonnell and Farrar
(1992) reported a reduction in cytochrome oxidase activity.
This coarse control of respiration involves regulation of
gene expression by carbohydrate availability. In tomato
fruits (Baldet et al., 2002) and roots (Devaux et al., 2003),
expression of some genes involved in the cell cycle and in
carbohydrate and nitrogen metabolism is quickly reduced
during the ®rst 2 d of darkness. Such an acclimation
occurred in our experiments after the 4 d pre-treatments. At
the beginning of the period of prolonged darkness, the total
carbohydrate content was reduced by 16 % in fruits 15 DAA
(about 30 % of the plant dry mass) and by 41±49 % in roots
and leaves (about 40 % of the plant dry mass) after low
compared with high carbon assimilation conditions. At the
same time, the plant respiration rate was 66 % lower after
low than after high carbon assimilation. This decrease in
respiration rate was more than proportional to the decrease
in carbohydrate content. Yet, at the organ or whole-plant
scale, respiration rate is related by a linear or saturation-type
curve to the carbohydrate content (e.g. Penning de Vries
434
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
F I G . 2. Time-course of soluble carbohydrate (circles) and starch (squares) concentrations during prolonged darkness and after 4 d of high (open
symbols) and low (closed symbols) carbon assimilation. Measurements were carried out on growing vegetative (young leaves, roots) and reproductive
organs [¯ower buds, fruits at the stage of cell division (5 DAA) and cell expansion (15 DAA)]. Mean 6 s.d. of eight analyses: two plants 3 two
extractions 3 two analyses.
et al., 1979; Gary, 1988b). Accordingly, Challa (1976)
characterized acclimation in vegetative cucumber plants by
a response curve of respiration rate to carbohydrate content
that was lower for plants grown under winter light than
under spring light conditions.
The decrease in starch and soluble carbohydrate concentrations observed in vegetative organs and in ¯owers
contrasts with their relative stability in fruits, irrespective
of their physiological stage. In roots, little starch was stored
and the pool of soluble carbohydrates decreased strongly
during the ®rst 24 h. However, Pressman et al. (1997) noted
that starch was stored in comparable concentrations to
soluble carbohydrates in roots of a different tomato cultivar
grown in Israel in winter. Indeed, Hewitt and Marrush
(1986) showed that there is strong genetic variability in the
starch±soluble carbohydrate balance in the vegetative
tissues of tomato plants. In leaves, starch is the most
variable carbohydrate pool (Bertin et al., 1999). Starch was
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
435
F I G . 3. Time-course of asparagine (triangles) and glutamine (diamonds) concentrations during prolonged darkness and after 4 d of high (open
symbols) and low (closed symbols) carbon assimilation. Measurements were carried out on growing vegetative (young leaves, roots) and reproductive
organs [¯ower buds, fruits at the stage of cell division (5 DAA) and cell expansion (15 DAA)]. Mean 6 s.d. of two plants.
rapidly exhausted following low carbon assimilation pretreatment but remained at high levels for 48 h after high
carbon assimilation pre-treatment. The availability of
carbohydrates was not estimated in stems and petioles.
These tissues mainly accumulate soluble carbohydrates
(Hewitt and Marrush, 1986). They are active sinks (Khan
and Sagar, 1966) and the amounts of carbohydrates they
store can be similar to those stored in leaves (lower in
Pressman et al., 1997; higher in Devaux, 2000).
A fairly stable concentration of soluble carbohydrates
was maintained in ¯owers throughout the period of darkness
after high carbon assimilation and for approx. 48 h after low
carbon assimilation. In the last experiment and after 48 h of
darkness, ¯owers certainly experienced a shortage of
assimilates that could lead to fruit set failure (Bertin,
1995). In contrast, during the cell division and cell
expansion stages, fruits could maintain their carbohydrate
concentration throughout the period of darkness, irrespective of the pre-treatment. Overall, leaves and stems were the
major sources of carbohydrates to support the growth of
fruits and maintain their carbohydrate content. Leaf samples
were harvested on the fourth and ®fth sympods. Given that
(1) concentrations of soluble carbohydrates vary little
among leaves; (2) concentrations of starch decrease to
zero on the second sympod (Bertin et al., 1999); and (3) the
carbohydrate content should be similar in leaves and stems,
a total content of carbohydrates available in the vegetative
tissues at the beginning of the period of darkness was
436
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
F I G . 4. Time-course of fruit (A) and stem (B) growth during prolonged
darkness and after 4 d of high (open symbols) and low (closed symbols)
carbon assimilation. Diameter expansion was measured every 5 min and
averaged on an hourly basis on six fruits and stems from six different
plants (mean 6 90 % con®dence interval).
estimated. It amounted to 16´6 and 5´8 g equivalent glucose
per plant after high and low carbon assimilation, respectively. During the next 72 h of darkness, 7´7 and 0´6 g
equivalent glucose per plant were respired in the same two
treatments. The difference between these two values is the
amount of carbohydrate available for the continuation of
growth in darkness: 8´9 and 5´2 g equivalent glucose per
plant, respectively. Analysis of the time-course of carbohydrate concentrations clearly indicates that under increasing
carbon starvation, fruits were given priority over vegetative
organs for the partitioning of assimilates. Such a priority for
reproductive organs was observed by Coggeshall and
Hodges (1980): shade treatments applied to soybean plants
reduced the starch concentration of all sink organs except
¯owers and pods.
Speci®c amino acids are considered to be indicators of
carbon stress. During the second stage of carbon starvation
when proteolysis occurs, nitrogen is stored in the form of
asparagine and glutamine, which limits the accumulation
of ammonium in cells (Brouquisse et al., 1992). However,
neither amino acid accumulated in roots despite the
apparent exhaustion of carbohydrates under prolonged
darkness. Asparagine and glutamine accumulated in leaves
but there were no clear differences related to the two pretreatments. Asparagine accumulated in ¯owers and glutamine in fruits during cell division. These results con®rm that
these indicators are not unequivocal. The next step of the
second stage of carbon starvation is the consumption of
glutamine and asparagine by the TCA cycle resulting in a
decrease in their concentration and in the production of
ammonium (Brouquisse et al., 1992). For the results to be
more conclusive, a larger variety of nitrogen compounds
(from proteins to ammonium) should have been analysed
and the expression of the related genes characterized.
Furthermore, the low concentrations of carbohydrates do
not necessarily mean that tissues have experienced carbon
starvation, provided that import of assimilates continues.
The permanent translocation of assimilates among organs in
the whole plant certainly attenuates the effects of carbon
starvation, that are more severe on excised organs (Baldet
et al., 2002).
The most striking result is the similarity of the timecourse of fruit expansion during prolonged darkness after
both carbon assimilation pre-treatments. Although carbohydrate reserves were reduced by 65 % after low carbon
assimilation, the fruit expansion rate was, on average, only
11 % lower throughout the period of darkness. In both
experiments, the fruit expansion rate started to decrease
sharply after 16 h of darkness, simultaneously with the
whole-plant respiration rate, as if growth substrates where
missing regardless of their availability at the beginning of
the period of darkness.
Two points can be made to explain this behaviour. First,
even after low carbon assimilation, suf®cient carbohydrate
was available to support fruit growth during 72 h of
darkness. The relative growth rate of fruit was estimated
from fruit expansion measurements by assuming that fruits
were spheres of 50 mm diameter at the beginning of the dark
period and had the same expansion rate in all three
dimensions. Tomato fruit dry matter is mainly constituted
by carbohydrates (Davies and Hobson, 1981). Its increase at
the whole-plant scale during 72 h of darkness was estimated
by hypothesizing that all fruits had the same exponential
growth curve (note that the fruit growth curve is actually
closer to a Gompertz function; Bertin, 1995) and, therefore,
the same relative growth rate. The amount of assimilates
dedicated to fruit growth was roughly estimated to be 8´7
and 4´3 g equivalent glucose per plant, after high and low
carbon assimilation, respectively; these values were still
lower than the amounts estimated to be available at the
beginning of the dark period.
A second point of discussion is that the expansion rate of
¯eshy fruits, such as those of tomato, depends more on
temperature and on plant water status than on the availability of assimilates (Pearce et al., 1993a, b). Grange and
Andrews (1995) reported that the respiration rate of young
tomato fruits is closely linked to the import rate of
assimilates and weakly related to the fruit expansion rate.
However, in the present experiments which took place in
growth chambers, plants experienced no changes in temperature and water status during the period of prolonged
darkness. In comparable controlled conditions, Devaux
(2000) observed no changes in the dry matter concentration
of tomato fruits over a 9-d period of darkness. It may be
hypothesized that the fruit expansion rates measured in the
present experiments re¯ect the import rates of assimilates.
Gary et al.ÐCarbon Balance of Tomato Plants in Prolonged Darkness
In a context of increasing carbon starvation at the wholeplant scale, the growth rate of fruits was maintained, as
indicated by the variations in fruit diameter, whereas the
growth of vegetative organs was impaired, as indicated by
the whole-plant respiration measurements and the variations
in stem diameter.
The sequence of events observed during prolonged
darkness can also be analysed in reference to the modelling
of the plant carbon balance. As stated in the Introduction,
the so-called maintenance status of plant metabolism should
only be a transient stage between the ®rst and second stages
of carbon starvation. Values of the maintenance respiration
rate at the whole-plant scale were estimated from maintenance coef®cients for leaf, stem, fruit and root tissues, and
acclimation of respiration to environmental conditions was
calculated through a correlation with relative growth rate
(Heuvelink, 1995). The average relative growth rate was
estimated from the net assimilation rate measured during the
4 d pre-treatment. The calculated maintenance respiration
rate was 1´125 and 0´300 mmol per plant s±1 after high and
low carbon assimilation, respectively. When these values
were compared with the time-course of total respiration rate
measured in expts 1 and 2 (Fig. 1), the maintenance stage
(corresponding to the cessation of whole-plant growth and
to the exhaustion of the carbohydrate pool) would have
occurred after about 24 h of darkness in the ®rst case and
after approx. 5 h in the second, according to the model of
Seginer et al. (1994).
The present analysis shows that modelling the carbon
balance at the whole-plant scale is an over-simpli®cation.
The shortage of carbohydrates leads to changes in the
carbon balance that differ in growing fruits and in vegetative
organs. However, it can be argued that the stage of
maintenance that was identi®ed by existing tomato crop
models (Seginer et al., 1994; Heuvelink, 1995) is not far
from the time of exhaustion of the carbohydrate reserves in
the vegetative organs. In fact, optimization of plant growth
should be based on the ability of the vegetative apparatus
not only to feed the organs of accumulation, but also to
maintain the integrity of its structures. In this respect, the
present results validate the existence of a lower threshold of
carbohydrate reserves and its use in optimization procedures
(Tchamitchian and Ioslovich, 2000).
ACKNOWLEDGEMENTS
We acknowledge the helpful technical assistance of B.
Brunel. This work was funded by grants from INRA (AIP
Agraf) and ReÂgion Aquitaine.
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