Sucrose cycling in heterotrophic plant cell metabolism:
first step towards an experimental model
Claude Roby, Sandra Cortès, Marina Gromova, Jean-Luc Le Bail, Justin K M Roberts*
1
CEA, CNRS and UJF, UMR 5019 Physiologie Cellulaire Végétale, 17 rue des Martyrs,
38054 Grenoble cedex 9, France.
*Department of Biochemistry, University of California, Riverside, CA 92521, USA
Corresponding author: Claude Roby, DRDC/PCV/RMBM, CEA, 17 rue des Martyrs, 38054
Grenoble cedex 9 France. Tel: 33 4 38 78 37 51 Fax: 33 4 38 78 54 83 Email: [email protected]
Abstract
Sucrose is the cornerstone of higher plant metabolism. Produced by photosynthesis,
sucrose is the main substrate for respiration and biosynthesis. The emerging idea is that
sucrose may act as regulator of its own metabolism, characterized in particular by a
permanent process of degradation and formation. This sucrose turnover may control several
important physiological functions. Of particular concern is an energy dependent cycle
involving the hexokinase. This report presents an experimental approach to define
quantitatively physiological states of suspension-cultured plant cells wih reference to their
sucrose content and respiration rate. Sucrose depletion of normal cells incubated in a medium
devoid of sugar is measured in vivo using 13C and respiration is simultaneously recorded.
Results obtained with sucrose-storing cells and Arabidopsis thaliana show that respiration
rate is closely linked to the available sucrose. Sucrose-depleted cells offer a stable model to
study the bioenergetics of the process.
Key words: Plant cells, Sucrose metabolism, Sucrose cycling, Bioenergetics.
Abbreviations: HXK, hexokinase (generic); NMR, nuclear magnetic resonance
Introduction
In normal conditions, sucrose is the main respiratory and growth substrate of higher
plants. Sucrose and starch are the end products of photosynthesis. Sucrose, formed in the leaf
cytosol, is transported through the phloem to the sink tissues where it is metabolized or stored
in the vacuole (Taiz & Zeiger, 1991). During the night, sucrose is formed from starch,
produced and stored in the chloroplasts during the day. Conversely, in non-photosynthetic
cells, starch can be formed into and stored in amyloplasts from sucrose (Fernie et al., 2002).
Sugar metabolism is also characterized by a continuous process of degradation and
biosynthesis of sucrose, observed in very different plant cells (Dancer et al., 1990; Wendler et
al., 1990; Geigenberger & Stitt, 1991 & 1993; Hill & ap Rees, 1994). This sucrose cycle
involves the invertase, hexokinase, hexose phosphate isomerase, UDPglucose
pyrophosphorylase, sucrose phosphate synthase and sucrose phosphate phosphatase. In
normal conditions, the flux of carbon cycling is high, and the energy cost induced by the ATP
consumption through HXK is a matter of debate (Dieuaide-Noubhani et al., 1995; Trethewey
et al., 1999). Futhermore, this apparent futile cycle could have major physiological functions
such as control of respiration (Dancer et al., 1990), maintenance of osmotic potential
(Geigenberger et al., 1997) control of sugar accumulation (Rohwer & Botha, 2001) and sugar
signaling (Cortès et al., 2002). Understanding the integration of the sucrose cycle into plant
energy metabolism still needs quantitative data. In this perspective, we started to set up an
experimental methodology to measure as directly as possible sucrose cycling fluxes as a
function of physiological parameters such as the amount of sucrose available to the cell
(endogenous or exogenous) and the energy status. The first step, presented here, was to define
quantitatively physiological states of reference with regard to sucrose reserve and respiration.
Materials and methods
Feast and famine in plant cells
In the life of a plant, sugar supply to the sink tissues varies considerably and several
physiological situations, such as the decrease in photosynthesis, can lead to sucrose
deprivation. Sugar-fed cells or cells containaing a high amount of sucrose define the normal
physiological state while sucrose-depleted cells without exogenous sugar are in a stressed
state. Gene expression is highly modulated by the carbohydrate status: "feast" genes are
enhanced (either expressed or repressed) by sugar abundance while "famine" genes are
enhanced (id) by sugar depletion (Koch, 1996). A lack of exogenous sugars strongly limits
respiration (Saglio & Pradet, 1980) which decreases during consumption of the sucrose and
starch reserves when no exogenous sugars are available (Journet et al., 1986; Brouquisse et
al., 1991). The total amounts of protein and fatty acid decrease later, following increase in
lypolysis (Dieuaide et al., 1992) and proteolysis (James et al., 1993). Intracellular autophagy
(Douce et al., 1988) develops on the long term, allowing the cell to cope reversibly with
carbon starvation. The physiological and metabolic response of sugar-deprived cells can be
monitored by the measurements of respiration rate, and accumulation of specific lipid
residues (Roby et al., 1987) and amino acids (Genix et al., 1990). Thus, a stressed state of
sucrose-depleted cells not yet fully engaged into autophagy can be defined precisely.
Suspension-cultured cells
Two cell lines cultivated in suspension with sucrose as the only carbon source were used.
They were subcultured three times at four days interval before experiments in the original
medium supplemented with 52 mM sucrose. Sycamore (Acer pseudoplatanus L.) cells, a
model for metabolic studies, accumulate large amounts of sucrose in the vacuole (Bligny,
1977). Very little is known about the metabolism of isolated cells of Arabidopsis thaliana.
(Axelos et al., 1992), the reference in plant genomics.
In vivo measurements
Cells were gently packed between two filters in an NMR tube and a nutrient medium of
controlled composition, temperature, pH and oxygen concentration was circulated through the
sample at high flow rate. Respiration rate was measured on line during in vivo NMR
measurements as explained Fig 1. Endogeneous sucrose concentration was determined from
the intensities of the CH resonances in 13C NMR spectra. The time courses of the most
abundant phosphorylated mobile metabolites were obtained from 31P NMR spectra.
Results
Sycamore cells
When the nutritive medium containing 52 mM sucrose was replaced by a medium without
sucrose the internal sucrose concentration decreased from about 60 mM to around 6 mM
within 20 h (Fig 1). The respiration rate decreased in a similar way, and both time courses
could be fitted with a third order polynomial function. A physiological state of sucrosedepleted cells was defined after 20 h of sucrose starvation for two reasons. First, sucrose
depletion is significant, but, according to the amount of significant metabolite markers,
intracellular macroautophagy is not engaged yet (Roby et al., 1987). Second, the amount of
residual sucrose is close to the threshold level needed for sucrose detection by natural
abundance 13C NMR. Below, sucrose enrichment following labeling from exogenous [1-13C]glucose, could not be measured to assess sucrose cycling in sucrose-depleted cells.
45
120
[Sucout] = 52 mM
[Sucout] = 0 mM
[Glcout] = 25 µM
100
35
-1
-1
Respiration rate (l ) (O2 µmol. h .g )
40
80
25
60
20
15
40
Internal sucrose (s ) (mM)
30
10
20
5
0
0
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Time (h)
Figure 1. Time courses of endogenous sucrose and respiration rate in sycamore cells without supply
of exogenous sucrose. Cells (9 g FW) were perifused during 4 h in the NMR tube with 52 mM
sucrose. A new medium devoid of sucrose replaced the initial one and at time zero the cells and the
perifusion system had been thoroughly rinsed. After 20 h of sucrose starvation, 25 mM of glucose was
added to the external medium. Respiration rate was obtained by measuring the partial oxygen pressure
in the medium before and after the sample with the same oxygen electrode (type 12/120 Mettler
Toledo, Paris, Fr) and amplifier (type PO2-NUM, Radiometer Analytical, Lyon, Fr) using a threevalve pneumatic bypasss commutated every 15 minutes. Interleaved 13C and 31P NMR spectra were
measured at 100. 6 and 162 MHz respectively using a Bruker AMX 400 spectrometer (Wissembourg,
France).
In this starvation stage, supplying 25 mM glucose to the external medium increased
slightly the respiration rate whithout significantly changing the amount of endogenous
sucrose. The respiratory response of the cells to the concentration of exogenous glucose can
been obtained this way.
Arabidopsis thaliana
In opposition to the sycamore cells response, the respiration rate of Arabidopsis thaliana
cells decreased abruptly after removal of the exogenous sucrose, but remained above 60% of
the control value for quite a long time (Fig 2). No sucrose could be detected in 13C NMR
spectra of living cells. Enzymatic analysis confirmed that Arabidopsis thaliana cells
cultivated with 52 mM sucrose did use up sucrose during cell division and growth but did not
store it. The concentration of sucrose in control cells was around 2 mM as compared to 60 and
100 mM in sycamore (Fig 1) and sugarcane (Wendler et al., 1990) cells respectively. Residual
amounts of sucrose after several hours starvation show that the time course of respiration rate
does not follow the one for sucrose. The nature of the substrate maintaining Arabidopsis
thaliana cells respiration in the absence of sucrose is presently under investigation.
The time courses of cytosolic phosphate, G6P and ATP pools, monitored step by step in
stressed cells during pulses external glucose could lead to a thorough study of the fast
transition observed from control to starvation, by using adequat glucose concentrations.
30
V O2 (µmol. h-1. g-1)
25
20
[ Suc IN ] = 0.7 mM
[ Suc IN ] = 2 mM
15
10
[ Suc IN ] = 0.3 mM
5
With Sucrose 52 mM
-6
-4
-2
Without Sucrose
0
2
4
6
8
10
12
14
Time (h)
Figure 2. Time course of the respiration rate of Arabidopsis thaliana cells with and without sucrose
supply. Cells (14 g FW) were perifused during 6 h in the NMR tube with 52 mM sucrose. At time zero
cells had been rinsed with a new medium devoid of sucrose. Respiration rate was measured as
indicated in Fig 1. Internal sucrose concentrations were determined this way: following NaOH
extraction and invertase action, glucose was measured using the G6P deshydrogenase-hexokinase
assay.
Discussion
Experimental methodology
Using suspension-cultured plant cells, an experimental methodology has been defined, in
which exogenous and endogenous sucrose amounts could be controled. The model so far
defined has a sound physiological background but the different biochemical time scales
present in this context of sugar starvation must be considered. Since biochemical adaptation to
this stress may imply a profound modification of the metabolic network, care will be taken to
consider what is happening at this level to interpret measurements of metabolite levels. To
quantify sucrose cycling in sucrose-depleted cells, metabolic labeling can be performed at any
time during the starvation process without significantly modifying the stressed state since the
new quasi steady-state induced by the glucose pulse is very close.
Sucrose-storing cells
In sycamore cells, respiration rate is sustained mainly by intracellular sucrose during the
first 10 h in a sucrose-deprived medium. Sucrose utilization is then progressively replaced by
starch breakdown but a sucrose pool is maintained for as long as 30 h. The autophagic activity
starts only when almost all the endogenous sucrose has disappeared. Therefore, within these
time limits, sucrose cycling can be studied as a function of the amount of endogenous sucrose
assuming that the metabolic network involved is not drastically modified. However, this
assertion should be checked by measurements of protein amounts and enzymatic activities.
Non sucrose-storing cells
Arabidopsis thaliana cells deprived of exogenous sucrose consume very quickly their
small sucrose reserve, and since they do not store much starch either, they may be engaged
early into intracellular autophagy. Therefore, 2D electrophoresis experiments are under way
to know if rapid changes occur in the enzyme amounts of the sucrose cycle. However,
because of the very low amount of sucrose stored, Arabidopsis thaliana cells constitute a neat
model to study glucose/sucrose utilization in newly alimented sugar-depleted heterotrophic
cells. Since Arabidopsis thaliana cells do not store sucrose, the experimental model set forth
could prove useful to study the related sugar sensing aspect of sugar metabolism. In
particular, it may be helpful in studying the HXK sensing hypothesis, and to find out if the
catalytic activity of HXK is tightly correlated or not to the signaling flux of the HXK
signaling pathway (Cortès et al., 2002).
Acknowledgements : Nathalie Pochon contributed skillfully to some NMR experiments
and Claire Desvignes participated enthousiastically to the enzymatic analysis of soluble
sugars. Discussions with Renaud Brouquisse on sugar signaling and sugar starvation were
very helpful for the development of the work which has been consistently encouraged by
Roland Douce. The referee kindly pointed out recent modelisation work on sucrose cycling.
References
Axelos, M., Curie, C., Mazzolini, L., Bardet, C. and Lescure, B., Plant Physiol. Biochem., 30
(1992) 123
Bligny, R., Plant Physiol., 59 (1977) 502
Brouquisse, R., James, F., Raymond, P. and Pradet, A., Plant Physiol., 96 (1991) 619
Cortès, S., Gromova, M., Evrard, A., Roby, C., Heyraud, A., Rolin, D., Raymond, P. and
Brouquisse, R., Plant Physiol., submitted (2002)
Dancer, J., Hatzfeld W.-D. and Stitt, M., Planta, 182 (1990) 223
Dieuaide, M., Brouquisse, R., Pradet, A. and Raymond, P., Plant Physiol., 99 (1992) 595
Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A. and Raymond, P., J. Biol. Chem.
270 (1995) 13147
Douce, R., Bligny, R., Dorne, A.-J. and Roby, C. Intracellular cannibalism in higher plant
cells, in Plant membranes: structure, assembly and function, The Biochemical Society,
London, GB., 1988, 189
Fernie, A. R., Willmitzer, L. and Trethewey, R. N., Trends Plant Sci., 7 (2002) 35
Geigenberger, P., Reimholz, R., Geiger, M., Merlo, L., Canale, V. and Stitt, M., Planta, 201
(1997) 502
Geigenberger, P. and Stitt, M., Planta, 185 (1991) 81
Geigenberger, P. and Stitt, M., Planta 189 (1993) 329
Genix, P., Bligny, R., Martin, J.-B., and Douce, R., Plant Physiol., 94 (1990) 717
Hill, S.A. and ap Rees, T., Planta, 192 (1994) 52
James, F., Brouquisse, R., Pradet, A. and Raymond, P., Plant Physiol. Biochem., 31 (1993)
845
Journet, E.-P., Bligny, R. and Douce, R., J. Biol. Chem., 261 (1986) 3193
Koch, K., Ann.Rev. Plant Physiol. Plant Mol. Biol., 47 (1996) 509
Roby, C., Martin, J.-B., Bligny, R., and Douce, R., J. Biol. Chem., 262 (1987) 5000
Rohwer, J. and Botha, F. C., Biochem. J., 358 (2001) 437
Saglio, P.H. and Pradet, A., Plant Physiol., 66 (1980) 516
Taiz, L. and Zeiger, E. Plant physiology, The Benjamin/Cummings Publishing Company,
Inc., Redwood city, California, 1991, 219
Trethewey, R.N., Riesmeier, J.W., Willmitzer, L., Stitt, M. and Geigenberger, P., Planta, 208
(1999) 227
Wendler, R., Veith, R., Dancer, J., Stitt, M. and Komor, E., Planta, 183 (1990) 31