Plant Physiol. (1998) 118: 1525–1532
Carbohydrates in Individual Cells of Epidermis, Mesophyll,
and Bundle Sheath in Barley Leaves with Changed Export or
Photosynthetic Rate1
Olga A. Koroleva*, John F. Farrar, A. Deri Tomos, and Christopher J. Pollock
School of Biological Sciences, University of Wales, Bangor, Gwynedd, Wales, United Kingdom (O.A.K., J.F.F.,
A.D.T.); and Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed, Wales,
United Kingdom (C.J.P.)
Carbohydrate metabolism of barley (Hordeum vulgare) leaves
induced to accumulate sucrose (Suc) and fructans was investigated
at the single-cell level using single-cell sampling and analysis. Cooling of the root and shoot apical meristem of barley plants led to the
accumulation of Suc and fructan in leaf tissue. Suc and fructan
accumulated in both mesophyll and parenchymatous bundle-sheath
(PBS) cells because of the reduced export of sugars from leaves
under cooling and to increased photosynthesis under high photon
fluence rates. The general trends of Suc and fructan accumulation
were similar for mesophyll and PBS cells. The fructan-to-Suc ratio
was higher for PBS cells than for mesophyll cells, suggesting that the
threshold Suc concentration needed for the initiation of fructan
synthesis was lower for PBS cells. Epidermal cells contained very
low concentrations of sugar throughout the cooling experiment.
The difference in Suc concentration between control and treated
plants was much less if compared at the single-cell level rather than
the whole-tissue level, suggesting that the vascular tissue contains a
significant proportion of total leaf Suc. We discuss the importance
of analyzing complex tissues at the resolution of individual cells to
assign molecular mechanisms to phenomena observed at the wholeplant level.
The major product of photosynthesis in temperate grass
leaves is Suc, which is stored, converted into fructan (the
main storage carbohydrate in the vegetative parts of coolseason grasses [Pollock and Cairns, 1991]), or transported
through the phloem to other parts of the plant. Fructan
synthesis in the leaves of temperate grasses is related to
high Suc concentrations in the tissue (Housley and Pollock,
1985; Cairns and Pollock, 1988); however, the control of the
competing processes of Suc export and fructan synthesis is
not yet clear. The enzymology of fructan synthesis in
grasses is also not completely understood (Pollock and
Cairns, 1991; Cairns, 1993, 1995).
Synthesis of fructan oligosaccharides in vitro requires
both high Suc and high fructosyltransferase concentrations
(Cairns 1992, 1995). This implies that the synthesis of fructan in vivo occurs in a cell compartment with high local
concentrations of both enzyme and substrate. The vacuole
is considered to be the site of fructan biosynthesis (Pollock
and Chatterton, 1988), but it is not clear whether the vacuoles of all cell types accumulate fructan equally. Therefore,
knowledge of the tissue localization of fructan synthesis
and accumulation is necessary to fully understand primary
carbohydrate metabolism in leaves. The SiCSA technique
(Tomos et al., 1994) has demonstrated that when barley
(Hordeum vulgare) leaves accumulate sugars they are not
uniformly accumulated in all cell types (Fricke et al., 1994;
Koroleva et al., 1997). The epidermis contains very low
concentrations of sugars and takes no part in carbohydrate
storage (Fricke et al., 1994; Koroleva et al., 1997).
A limitation of the SiCSA technique is that it is impossible to determine the proportion of sap from the vacuolar
and cytosolic compartments within any sample. The cytosol in mesophyll cells of wheat leaves occupies only 7.2%
of cell volume compared with 38.9% for vacuoles (Altus
and Canny, 1985). Cytosol occupies 6.7% of total cell volume in barley leaves (Winter et al., 1992). By contrast, the
vacuole occupies 99% of the whole cell volume in epidermal cells (Winter et al., 1992). The SiCSA data presented in
this study are therefore likely to represent vacuolar sugars
to a greater extent than those of the cytosol.
Cells of both the mesophyll and PBS in source leaves of
barley accumulate Suc (Koroleva et al., 1997) and are likely
to synthesize fructan. Although the mesophyll cells are the
major site of Suc synthesis, PBS cells are also capable of
photosynthesis, since they contain PSII activity and
Rubisco protein (Williams et al., 1989; O.A. Koroleva, unpublished data). The lateral cells of the PBS (S-type cells)
may also play an important role in the transport of assimilate to the phloem (Williams et al., 1989).
In this study we sought to determine whether individual
epidermal, mesophyll, and PBS cells have different quantitative and qualitative patterns of carbohydrate accumulation in barley leaves when carbohydrate accumulation in
the leaf was increased either by reducing export of Suc
from the leaf or by increasing photosynthetic rate. Cooling
of sinks (roots and shoot apical meristem) to 10°C reduced
Suc export from leaves and resulted in accumulation of
1
This study was supported by the Biotechnology and Biological
Science Research Council (research grant no. 5/PO 2288).
* Corresponding author; e-mail [email protected]; fax
44 –1–248 –370731.
Abbreviations: DP, degree of polymerization; PBS, parenchymatous bundle sheath; SiCSA, single-cell sampling and analysis; SST,
Suc-to-Suc-fructosyltransferase.
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1526
Koroleva et al.
total carbohydrate and in the induction of fructan synthesis
in leaves (Smouter and Simpson, 1991; Plum and Farrar,
1996; Koroleva et al., 1997). The carbohydrate content of
source leaves can also be increased by a higher rate of
photosynthesis without restricting the export of sugars;
therefore, we grew plants for 2 d under high photon fluence rates.
MATERIALS AND METHODS
Growth of Plants
Barley (Hordeum vulgare L. cv Klaxon) plants were grown
hydroponically in controlled-environment cabinets (Sanyo
Gallenkamp, Loughborough, Leicester, UK) at 20°C, a 16-h
light/8-h dark cycle, a photon fluence rate of 500 mmol
photons m22 s21, and 0.035% CO2. The CO2 concentration
was maintained by pumping air from 15 m above the
ground through the chamber, with four air changes per
hour. Seeds were soaked overnight and germinated for 4 d
on moist paper. Fourteen seedlings were then transferred
to a trough containing one-half-strength Long-Ashton nutrient solution (Hewitt, 1966). Solutions were changed
twice a week. The third leaf was fully expanded at about d
14 after transfer. All samples were taken from the middle
parts of the third leaves 1 to 5 d after full expansion of the
leaf blade.
Cooling Experiments
A low temperature was used to induce the accumulation
of fructan (Smouter and Simpson, 1991; Koroleva et al.,
1997). The plant roots and the lower 3 cm of the shoot
(including the shoot apex) were submerged in nutrient
solution at 10°C. The rest of the plant was kept at 20°C.
Cooling started on d 1 after the third leaf had fully expanded. Cooling continued for 4 d, and then the solution
was warmed to 20°C for the rest of the experiment. Parallel
sets of control plants were sampled at the same age as
cooled plants.
Plant Physiol. Vol. 118, 1998
detected with a refractive index detector (model RID-6A,
Shimadzu, Kyoto, Japan) and analyzed with integration
software (Valuchrom, Bio-Rad). The fractions were quantified using external standards of similar retention times.
Extraction of Sap from Individual Cells
Sap was extracted from individual epidermal, mesophyll, and PBS cells of barley leaves using a microcapillary.
The tip was inserted through a stomatal pore to reach
mesophyll and PBS cells. Details of the procedure were
given by Koroleva et al. (1997).
Hydrolysis of Fructans
Conventional acid hydrolysis cannot be used to hydrolyze fructans in single-cell samples because of their small
volumes (20–100 pL). We used enzymatic hydrolysis of
fructan polymers to Fru for subsequent analysis. Commercial yeast b-fructosidase (invertase) hydrolyzes the three
common fructan trisaccharides and also larger oligofructans (Cairns, 1993; Simpson and Bonnett, 1993), whereas
Suc phosphorylase will hydrolyze only Suc. We used TLC
(Cairns and Pollock, 1988) to assess the invertase activity
on fructans extracted from barley leaves. Both Suc and
fructan oligosaccharides up to DP 8 from barley leaves (Fig.
1, lanes 3 and 4) were hydrolyzed completely to hexoses by
yeast invertase at pH 4.6 within 1 h (Fig. 1, lanes 5 and 6).
At pH 7.5 invertase completely hydrolyzed the ethanolsoluble fraction (Fig. 1, lane 7) but not the water-soluble
fraction (Fig. 1, lane 8). Suc phosphorylase hydrolyzed
more than 50% of Suc during the same 1-h period (Fig. 1,
High Photon Fluence Rate
At full expansion of the third leaf, plants were transferred for 2 d to a chamber with photon fluence rate of 1000
mmol photons m22 s21. The photoperiod, temperature, and
CO2 concentration were unchanged.
Measurement of Carbohydrates in Leaf Tissue
Tissue samples were taken from the middle part of leaf
lamina, killed in boiling 90% ethanol, and extracted in 90%
ethanol at 60°C (Koroleva et al., 1997). Ethanol-soluble
sugars were analyzed by HPLC (Cairns and Pollock, 1988).
The ethanol extracts were combined, evaporated, and redissolved in water. This fraction was deionized using
Dowex 50 (H1) and Dowex 1 (CO322) columns, concentrated, filtered through a 0.45-mm polysulfene membrane,
and analyzed (Aminex HPX 87C column, Bio-Rad) at 85°C,
with water as the solvent. The individual fractions were
Figure 1. Specificity of invertase and Suc phosphorylase activity.
TLC plate with lanes containing barley fructans from 80% ethanolsoluble (lane 3) and water-soluble (lane 4) fractions and products of
their hydrolysis by yeast invertase (Sigma) at pH 4.6 (lanes 5 and 6,
respectively) and at pH 7.5 (lanes 7 and 8, respectively). Suc phosphorylase hydrolyzed Suc from the same water-soluble fraction (lane
9). Oligofructans from the ethanol-soluble fraction were stable in the
absence of enzymes at pH 4.6 (lane 10). Suc and Fru (lanes 1 and 11,
respectively) and oligoinulins from Helianthus tuberosus (lanes 2 and
12) were used as markers.
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Carbohydrates in Barley Epidermal, Mesophyll, and Bundle-Sheath Cells
1527
lane 9). The ethanol-soluble fraction (Fig. 1, lane 3) was
stable at pH 4.6 (Fig. 1, lane 10).
The amount of fructan oligosaccharides present in samples was determined as the difference between the amount
of hexose produced by the two enzymes (invertase and Suc
phosphorylase). The carbohydrate-to-enzyme concentration ratios used for TLC assay were much higher than for
single-cell assays, in which complete hydrolysis was always ensured.
Enzymatic Microassay of Carbohydrate Concentrations
Concentrations of various carbohydrates in the sap from
single cells were measured using a microfluorometric assay. This assay involves enzymatic dehydrogenation of
Glc-6-P derived sequentially from Glc, Fru, Suc, and fructans, with a corresponding reduction of NADP to NADPH.
A microscope photometer (MPV Compact 2 Fluorovert,
Leitz, Wetzlar, Germany) fitted with filter block A and
software (Leitz) was used to measure the fluorescence of 4to 5-nL droplets of a reaction mixture placed on a microscope slide inside a 4-mm-deep aluminum ring under 3
mm of water-saturated paraffin oil. The reaction mixture
contained 68 mm imidazole buffer, pH 7.5, 5.6 mm MgCl2,
5.6 mm ATP, 0.1% BSA, 23.8 mm NADP, and 1.2 mm
KH2PO4. Three identical subsets of droplets were placed on
the slide. The standards and samples (approximately 10
pL) were added with a constriction pipette in the same
sequence for all three subsets. Then, 100 pL of Suc phosphorylase (10 units/mL; Sigma) was added to all droplets
in subset 2, and 100 pL of b-fructosidase (invertase; 1330
units/mL; Boehringer Mannheim) was added to all droplets in subset 3.
Excess invertase was used, since the enzyme must operate at nonoptimal pH. The initial fluorescence was recorded for all droplets, and Glc phosphorylation was
started in all subsets by adding approximately 100 pL of a
hexokinase/Glc-6-P dehydrogenase (Boehringer Mannheim) mixture (85/43 units/mL). After 10 to 15 min the
reaction was complete (the fluorescence readings increased
and then became stable), with the change in NADPH fluorescence being proportional to the amount of Glc in the
droplet. The second step of the assay was started by adding
100 pL of phosphoglucose isomerase (Boehringer Mannheim; 175 units/mL). The additional change in NADPH
fluorescence after 20 to 30 min indicated the amount of Fru.
Measurements of subset 1 gave values for Glc and Fru
concentrations present initially in the samples and standards, subset 2 gave the initial amounts of Glc and Fru plus
Fru derived from Suc by Suc phosphorylase (the second
molecule produced in this reaction, Glc-1-P, is not a substrate for further reactions), and subset 3 was used to
estimate total hexose content in the samples and standards
after complete hydrolysis of both Suc and fructans. This
method provides the total amount of fructan but no information about the DP. Therefore, all measurements of fructan from single cells are expressed as hexose units.
Figure 2. Time course of the content of carbohydrates analyzed by
HPLC in tissue samples from the third leaves of barley plants with
their roots and shoot apical meristem cooled (A) or uncooled (B;
controls). Each point is an average 6 SD of three plants. C, Suc and
total hexose units from fructan shown on a molar basis to allow
comparison with single-cell fructan measurements, in which the DP
was unknown. f.w., Fresh weight.
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Koroleva et al.
Plant Physiol. Vol. 118, 1998
RESULTS
Carbohydrates from Whole-Leaf Tissue
Cooling of sinks increased the ethanol-soluble fraction
(which contains most of the carbohydrate) of total carbohydrate of the whole-tissue extracts (Fig. 2), mainly because of increased Suc and fructan concentrations (DP 4 1
5). For example, the Suc concentration was much higher at
the end of the third photoperiod (60 h) in the leaf tissue of
cooled plants, i.e. approximately 30 mg g21 fresh weight
(Fig. 2A) compared with 13 mg g21 fresh weight in control
plants (Fig. 2B).
Total carbohydrate accumulation would be somewhat
higher than the above values (Koroleva et al., 1997). Hexoses (Glc and Fru) remained at very low concentrations.
Suc concentration followed a diurnal pattern in both cooled
(Fig. 2A) and control (Fig. 2B) plants. Only part of one cycle
is illustrated for the control plants. At the end of the third
photoperiod (60 h), the concentrations in control plants
were similar compared with the plants at the beginning of
the experiment.
The concentration of fructan trisaccharide fluctuated
only slightly and remained quite low, which is typical
behavior for an intermediate product. The concentration of
fructan with a DP 4 1 5 increased steadily during cooling
but decreased abruptly immediately after the plants were
returned to control conditions (Fig. 2A). To allow comparison of HPLC and SiCSA results, Figure 2C presents an
estimate of the molar concentration for Suc and hexose
derived from fructan from Figure 2A. For this estimation it
was assumed that 1 kg of tissue was equivalent to 1 L of
solution.
Carbohydrates from Single Cells
Epidermal cells contain negligible concentrations of sugars and do not accumulate additional carbohydrate when
sinks were cooled (Fig. 3A) or after exposure to a high
photon fluence rate (data not shown).
In both mesophyll and PBS cells of control plants, Suc
and fructan concentrations increased with leaf age between
d 1 (1 d after full expansion of the leaf blade) and d 4 (Fig.
3, B and C). In plants cooled between d 1 and 4, fructan
accumulation was 4 to 6 times higher in mesophyll and PBS
cells than in control plants. However, the mesophyll Suc
concentration was not different for cooled and control
plants (Fig. 3B). In PBS cells of the cooled plants the average concentration of Suc after 84 h of cooling was 3 times
higher than in the control plants at the same time (Fig. 3C).
High light also stimulated fructan synthesis (Fig. 4). In any
single experiment (e.g. Fig. 3 or 4), the fructan-to-Suc ratio
(in millimolar) was always higher in PBS cells than in
mesophyll cells.
Transferring plants to high light for 2 d had an effect
similar to that of cooling. Fructan synthesis was induced,
but the total amount of sugars that accumulated was much
higher in PBS cells than in mesophyll cells (because of the
higher fructan concentration; Fig. 4). The apparent threshold Suc concentration for initiation of fructan synthesis was
Figure 3. Concentrations of Glc, Fru, Suc, and fructan (as hexose
units) in individual epidermal (A), mesophyll (B), and PBS (C) cells
from plants with roots and shoot apical meristem cooled to 10°C and
in leaves of control barley plants. Treated plants were cooled for 3 (A)
and 4 d (B and C). Control plants were not cooled. Each bar is an
average 6 SD for three plants; two or three samples of each cell type
were analyzed from each plant.
approximately 100 mm for PBS cells. In mesophyll cells a
fixed threshold was less apparent as Suc increased to more
than 200 mm at the end of d 1 and then declined to 100 mm
by the end of d 2, possibly due to fructan synthesis.
Data from more than 10 experiments were pooled (Fig. 5)
to show the trends for each sugar during the 88 h of cooling
and subsequent rewarming during a 5-d experiment. Free
Glc and Fru concentrations were usually less than 20 mm in
both mesophyll and PBS cells for cooled and control plants
(Fig. 5, A, B, F, and G), although Glc peaked by 60 h.
Fructan and total carbohydrate concentration were
broadly similar for mesophyll and PBS cells in the cooling
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Carbohydrates in Barley Epidermal, Mesophyll, and Bundle-Sheath Cells
Figure 4. Concentrations of Glc, Suc, and fructan (as hexose units) in
individual mesophyll (A) and PBS cells (B) in barley plants grown at
500 mmol photons m22 s21 before transfer to high-light conditions
(1000 mmol photons m22 s21) and 1 and 2 d after. Each bar is an
average 6 SD for three plants.
experiment, although fructan accumulation may have
started earlier in PBS cells (Fig. 5, D, E, I, and J). The
fructan-to-Suc ratio was much higher for PBS cells than for
mesophyll cells, and they were both higher than the ratio
calculated from HPLC data for whole-leaf tissue (Fig. 6).
DISCUSSION
SiCSA showed that mesophyll and PBS cells both accumulate Suc and fructans to high concentrations, especially
under conditions of reduced export or increased photosynthetic rate. It is interesting that the epidermis does not act
as a buffer for leaf Suc accumulation.
High light and sink cooling resulted in quantitatively
similar responses, suggesting that the secondary effects of
root cooling, such as root-to-shoot signaling or a change in
the root hydraulic conductivity, do not affect the accumulation of sugars induced by low sink activity.
The SiCSA technique as described here cannot distinguish between vacuolar and cytoplasmic compartments. If
we assume that the ratio of vacuolar sap and cytosol in
single-cell samples is the same as for intact cells, the samples represent more of the vacuole (which occupies 38.9%
to 73% of mesophyll and 99% of epidermal cell volume)
than that of the cytosol (6.7% to 7.2% for mesophyll), and
the chloroplasts occupy 17% to 19% of the cell volume in
the mesophyll (Altus and Canny, 1985; Winter et al., 1993).
Because the Suc concentration in the mesophyll was
nearly the same for plants at the end of the light period
irrespective of the treatment (control, reduced leaf export,
or increased photosynthetic rate), there may be control of
1529
the upper level of Suc accumulation. One explanation is
that there is a definite threshold in Suc concentration (150–
200 mm) for the induction of fructan synthesis (Pollock,
1986).
The existence of a threshold concentration of Suc at
which fructan synthesis is induced is consistent with findings from other studies. Illuminated, excised leaves of Lolium temulentum L. provided an estimate of 15 mg g21 fresh
weight (Cairns and Pollock, 1988), excised illuminated barley leaves had 18 to 20 mg g21 fresh weight (Tomos et al.,
1992; Simmen et al., 1993), and excised barley leaves with
Suc applied exogenously in the dark suggested a value of
17 mg g21 fresh weight (Wagner et al., 1986). These values
all fall within a range of 40 to 60 mm if expressed on a
whole-tissue basis. Subsequent fructan synthesis appears
to require this range of Suc concentration in leaf tissue
(Wagner et al., 1986; Cairns and Pollock, 1988; Cairns,
1992).
Suc concentrations similar to those presented here were
found in isolated mesophyll protoplasts from illuminated,
excised leaves of L. temulentum (Cairns et al., 1989). These
were 110 mm (assuming that all Suc is vacuolar) or 100 mm
(assuming equal concentrations in the cytoplasm and vacuole). In vitro Km values for enzymes of fructan synthesis
in grasses suggest that such high Suc concentrations are
needed. The total SST activity increased linearly with substrate concentration over the range of 100 to 700 mm
(Cairns et al., 1989; Cairns, 1995). The activity of 1-SST from
excised barley leaves displayed a Km of 200 mm, whereas
6-SST activity increased linearly up to at least 600 mm Suc
(Simmen et al., 1993). In vitro Suc concentrations of less
than 80 mm led to synthesis of only the trisaccharide
isokestose.
The role and anatomical position of PBS cells were quite
different from those of mesophyll cells. The function of PBS
cells may be to accumulate Suc coming apoplastically or
symplastically from mesophyll cells and to regulate passage of this Suc to the interior of the vascular bundle for
export into the phloem. When export of Suc from the PBS
was reduced (Fig. 3) or its synthesis was increased (Fig. 4),
PBS cells accumulated up to approximately 100 mm Suc.
The concentration of Suc was usually lower in the PBS than
in the mesophyll, possibly because PBS cells start fructan
synthesis earlier (Figs. 3, 4, and 5, D and I). In PBS cells
(Fig. 3C) the threshold Suc concentration for the initiation
of fructan synthesis seems to be lower. Both the rate of Suc
and fructan accumulation and the fructan-to-Suc ratio are
different for mesophyll and PBS cells from the same leaf
(Figs. 3, 4, 5, C, D, H, and I, and 6).
The fructan-to-Suc ratio (Fig. 6) was much higher for PBS
cells; therefore, the rate of Suc export from these cells to the
veins may have been higher than that from mesophyll cells
to PBS. The equilibrium between Suc export and its use for
fructan synthesis may also be different to maintain a step
gradient in Suc concentration to allow continued diffusive
flow toward the PBS. The passage of Suc can proceed
through the plasmodesmatal continuity (Farrar et al., 1992).
The general behavior of PBS and mesophyll cells in terms
of Suc accumulation and fructan synthesis are, however,
broadly similar.
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Koroleva et al.
Plant Physiol. Vol. 118, 1998
Figure 5. Time courses of different carbohydrate concentrations during cooling experiments in mesophyll cells (A-E) and
PBS cells (F-J) of barley plants. Each point is an average 6 SD for three to 10 plants combined from 11 experiments. The line
was fitted by hand to show the general trend.
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Carbohydrates in Barley Epidermal, Mesophyll, and Bundle-Sheath Cells
Figure 6. Change of the fructan-to-Suc ratio expressed in millimolar
during the cooling experiment for mesophyll and PBS cells and for
whole-leaf tissue. Data from Figures 2 and 5 were used for the
calculation of these values.
The concentration of Glc was always at least 10 times less
than that of Suc in whole-leaf extracts (Fig. 2). However, it
was much higher in some cell-sap samples, with a peak Glc
concentration of more than 50% of the Suc concentration
appearing at d 3 in both mesophyll and PBS cells (Fig. 5, A
and F). This Glc peak is also seen in Figure 2 but is not as
highly expressed. The increase in Glc concentration following induction of fructan synthesis has also been seen in
excised primary barley leaves (Wagner et al., 1986; Simmen
et al., 1993) at values up to 55 mm averaged for the leaf
tissue. This Glc concentration was not due to invertase
activity, because there was no accumulation of Fru at the
same time. Furthermore, the activity of acid invertase in the
mesophyll and PBS cell sap was low (Koroleva et al., 1997).
It may be that a high Suc concentration, together with
activity of specific fructosyltransferases that use the fructosyl moiety from the Suc molecules to build up fructan
polymers, results in the accumulation of Glc residues. This
accumulation of Glc could also be responsible for the decrease in the CO2 assimilation rate (Krapp et al., 1991) that
is observed in the leaves of barley plants during cooling
(Plum and Farrar, 1996; Koroleva et al., 1997).
Sugars control the expression of many plant genes and
therefore many metabolic and developmental processes
(Koch, 1996). If sugar repression of photosynthesis represents a general mechanism for the regulation of the photosynthetic rate in response to changes in demand (Jang and
Sheen, 1994), Suc is not likely to be the major agent since its
concentration in mesophyll cell sap is generally within the
range of that in control plants. Jang and Sheen (1994)
showed in maize that the expression of photosynthetic
genes was suppressed by lower concentrations of Glc than
of Suc, suggesting that the ability of Suc to cause repression
is dependent on its hydrolysis. Glc has been shown to act as
a metabolic regulator independently of stress-related stimuli (Ehness et al., 1997).
1531
Comparing sugar concentrations measured at the tissue
level and in single mesophyll and PBS cells gives a unique
opportunity to compare carbohydrate accumulation in a
whole leaf with processes happening in individual cells.
Comparison of the fructan-to-Suc ratio of single cells with
that for whole tissue suggests that a substantial part of the
leaf Suc was located in cells that were not sampled, most
likely the vascular tissue. Indeed, autoradiography of barley leaves fed 14CO2 shows strong labeling of the veins (J.
Farrar, unpublished data).
It was not possible earlier to conclude from tissue-level
measurements the actual concentration of Suc in leaf cells.
For Suc content measured in whole tissue it is especially
important to know what proportion of it is located in the
phloem, where the concentration is extremely high. This
was estimated by Winter et al. (1992) to be 1030 mm at 9 h
into the light period and 930 mm after 5 h in the dark,
whereas the Suc concentration in the cytosolic compartment of the leaf (9 h into the photoperiod) was calculated
as 150 mm. The total volume of sieve tube and companion
cells is unknown, and it is not possible to compare the
amounts of Suc located in the leaf cells and the phloem
elements.
We recalculated the expected amounts of carbohydrate
in various leaf compartments from our single-cell data
using cellular volumes estimated by Winter et al. (1993) for
primary barley leaves: 27% in the epidermis, 42% in the
mesophyll, and 6% in the veins. Similar values were obtained for seven annual and perennial grass species (Garnier and Laurent, 1994). Because epidermal cells contain a
negligible concentration of Suc, the concentration of about
100 mm in mesophyll cells would correspond to 42 to 62
mm Suc in whole-leaf tissue. The measured tissue concentrations are higher, i.e up to 80 mm (Fig. 2C). The difference
in concentration of at least 20 mm is consistent with high
concentrations in the phloem, the volume of which is less
than the 6% found in the veins (Winter et al., 1993; Garnier
and Laurent, 1994). However, calculating the concentrations for a volume of 6% gives values of approximately 330
mm, which is one-third of the estimated value of the
phloem Suc concentration (1 m; Winter et al., 1992). If
phloem elements occupy one-third of the vascular tissue,
they would contain approximately 1 m Suc. These calculations are supported by the fact that the fructan concentration, which presumably is excluded from bundles, was
lower at the tissue level (Fig. 2C) than in mesophyll and
PBS cells.
It is clear that metabolism is compartmented between
subcellular organelles, and much is known of the subcellular organization of photosynthesis. Until now, however,
it has proved very difficult to assess the intercellular compartmentation within the same subcellular compartment.
This study shows that different cells within a complex
tissue such as a leaf behave very differently. Applying the
SiCSA technique will make it considerably easier to assign
molecular mechanisms to phenomena that until now have
been accessible only at the whole-leaf level.
Received June 3, 1998; accepted September 8, 1998.
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1532
Koroleva et al.
LITERATURE CITED
Altus DP, Canny MJ (1985) Loading of assimilates in wheat
leaves. II. The path from chloroplast to vein. Plant Cell Environ
8: 275–285
Cairns AJ (1992) Fructan biosynthesis in excised leaves of Lolium
temulentum L. V. Enzymatic de novo synthesis of large fructans
from sucrose. New Phytol 122: 253–259
Cairns AJ (1993) Evidence for the de novo synthesis of fructan by
enzymes from higher plants: a reappraisal of the SST/FFT
model. New Phytol 123: 15–24
Cairns AJ (1995) Effects of enzyme concentration on oligofructan
synthesis from sucrose. Phytochemistry 40: 705–708
Cairns AJ, Pollock CJ (1988) Fructan biosynthesis in excised leaves
of Lolium temulentum L. I. Chromatographic characterisation of
oligofructans and their labelling patterns following 14CO2 feeding. New Phytol 109: 399–405
Cairns AJ, Winters A, Pollock CJ (1989) Fructan biosynthesis in
excised leaves of Lolium temulentum L. III. A comparison of the
in vitro properties of fructosyl transferase activities with the
characteristics of in vivo fructan accumulation. New Phytol 112:
343–352
Ehness R, Ecker M, Godt DE, Roitsch T (1997) Glucose and stress
independently regulate source and sink metabolism and defense
mechanisms via signal transduction pathways involving protein
phosphorylation. Plant Cell 9: 1825–1841
Farrar J, van der Shoot C, Drent P, van Bel A (1992) Symplastic
transport of Lucifer Yellow in mature leaf blades of barley:
potential mesophyll-to-sieve-tube transfer. New Phytol 120:
191–196
Fricke W, Leigh RA, Tomos AD (1994) Concentrations of inorganic and organic solutes in extracts from individual epidermal,
mesophyll and bundle-sheath cells of barley leaves. Planta 192:
310–316
Garnier E, Laurent G (1994) Leaf anatomy, specific mass and
water content in congeneric annual and perennial grass species.
New Phytol 128: 725–736
Hewitt EJ (1966) Sand and water culture methods used in the
study of plant nutrition. In Technical Communication no. 22.
Commonwealth Agricultural Bureaux, Farnham Royal, UK
Housley TL, Pollock CJ (1985) Photosynthesis and carbohydrate
metabolism in detached leaves of Lolium temulentum L. New
Phytol 99: 499–507
Jang JC, Sheen J (1994) Sugar sensing in higher plants. Plant Cell
6: 1665–1679
Koch KE (1996) Carbohydrate-modulated gene expression in
plants. Annu Rev Plant Physiol Plant Mol Biol 47: 509–540
Plant Physiol. Vol. 118, 1998
Koroleva OA, Farrar JF, Tomos AD, Pollock CJ (1997) Solute
patterns in individual mesophyll, PBS and epidermal cells of
barley leaves induced to accumulate carbohydrate. New Phytol
136: 97–104
Krapp A, Quick WP, Stitt M (1991) Ribulose-1,5-bisphosphate
carboxylase/oxygenase, other photosynthetic enzymes and
chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream. Planta 186: 58–69
Plum SA, Farrar JF (1996) Carbon partitioning in barley following manipulation of source and sink. Aspects Appl Biol 45:
177–180
Pollock CJ (1986) Fructans and the metabolism of sucrose in
higher plants. New Phytol 104: 1–24
Pollock CJ, Cairns AJ (1991) Fructan metabolism in grasses and
cereals. Annu Rev Plant Physiol Plant Mol Biol 42: 77–101
Pollock CJ, Chatterton NJ (1988) Fructans. In J Preiss, ed, The
Biochemistry of Plants, A Comprehensive Treatise, Vol 14: Carbohydrates. Academic Press, San Diego, CA, pp 109–140
Simmen U, Obenland D, Boller T, Wiemken A (1993) Fructan
synthesis in excised barley leaves. Plant Physiol 101: 459–468
Simpson RJ, Bonnett GD (1993) Fructan exohydrolase from
grasses. New Phytol 123: 453–469
Smouter H, Simpson R (1991) Fructan metabolism in leaves of
Lolium rigidum Gaudin. I. Synthesis of fructan. New Phytol 119:
509–516
Tomos AD, Hinde P, Richardson P, Pritchard J, Fricke W (1994)
Microsampling and measurement of solutes in single cells. In N
Harris, KJ Oparka, eds, Plant Cell Biology—A Practical Approach. IRL Press, Oxford, UK, pp 297–314
Tomos AD, Leigh RA, Palta JA, Williams JHH (1992) Sucrose and
cell water relations. In CJ Pollock, JF Farrar, AJ Gordon, eds,
Carbon Partitioning within and between Organisms. BIOS, Oxford, UK, pp 71–89
Wagner W, Wiemken A, Matile P (1986) Regulation of fructan
metabolism in leaves of barley (Hordeum vulgare L. cv Gerbel).
Plant Physiol 81: 444–447
Williams ML, Farrar JF, Pollock CJ (1989) Cell specialization
within the parenchymatous bundle sheath of barley. Plant Cell
Environ 12: 909–918
Winter H, Lohaus G, Heldt HW (1992) Phloem transport of amino
acids in relation to their cytosolic levels in barley leaves. Plant
Physiol 99: 996–1004
Winter H, Robinson DG, Heldt HW (1993) Subcellular volumes
and metabolite concentrations in barley leaves. Planta 191:
180–190
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