Journal of Experimental Botany, Vol. 51, No. 344, pp. 567–577, March 2000
Spatial and temporal distribution of solutes in the
developing carrot taproot measured at single-cell
resolution
Andrey V. Korolev1,3, A. Deri Tomos1, Richard Bowtell2 and John F. Farrar1
1 Ysgol Gwyddorau Biolegol, Prifysgol Cymru Bangor, Bangor, Gwynedd LL57 2UW, UK
2 Magnetic Resonance Centre, Department of Physics, University of Nottingham, Nottingham NG7 2RD, UK
Received 18 June 1999; Accepted 12 November 1999
Abstract
The time-course and spatial distribution of sugars and
ions in carrot (Daucus carota L.) was studied at fine
resolution using single cell (SiCSA) and tissue analysis.
Four phases of osmolyte accumulation in the taproot
were identified: an amino acid (germination) phase,
when internal sources of amino acids provide seedlings with osmotica; an ion phase, when inorganic and
organic ions were the main solutes; a hexose phase,
when concentrations of glucose and fructose sharply
increased and reached their maximum; and a sucrose
phase, when sucrose became the major solute. Spatial
distribution of sugar in taproot cells showed a general
trend of highest concentration on both sides of the
vascular cambium (some 200 mM sucrose, 150 mM
glucose) and a minimum in the pith (some 100 mM
sucrose, 60 mM glucose) and in periderm. Electrolytes
(e.g. potassium) followed a distribution generally reciprocal to that of sugars; minimum in the tissue adjacent
to the cambium (some 10 mM) and maximum in the
pith and periderm (some 60–100 mM). The cambial
cells contained unexpectedly low concentrations of
sugars and potassium. These spatial and temporal patterns indicate that amino acids, other electrolytes and
sugars are interchangeable in the tissue osmotic balance. The nature of the solute is developmentally
determined both temporally and spatially. During the
accumulation of electrolytes following the initial amino
acid phase, osmotic pressure to 420 mosmol kg−1
rises and then remains constant despite large changes
in the concentration of individual solutes. This indicates that osmotic pressure is regulated independently of the individual concentrations of solutes.
Key words: Daucus carota, solute accumulation, sugar
storage, SiCSA.
Introduction
Anatomical development of the carrot plant has been
well-documented (Havis, 1939; Esau, 1940). Formation
of the taproot secondary structure starts with meristematic activity between the primary xylem and phloem.
Towards the end of the first month after sowing, a
complete cambial sheath forms around the central primary xylem. The cambium produces phloem tissue to the
outside and xylem tissue to the inside. The secondary
phloem zone is covered by periderm, the taproot protective layer, and contains phloem elements and cells of
phloem parenchyma. The structure of the periderm and
outermost layer of the phloem zone may be closely related
with taproot resistance to splitting (McGarry, 1995). The
innermost layer of the phloem zone is assumed to contain
a high proportion of active phloem elements ( Esau, 1940;
Caplin, 1973) and long-distance transport of sucrose from
leaves to taproot occurs mainly here (Caplin, 1973;
Patrick, 1997).
The xylem zone contains xylem vessels and cells of
xylem parenchyma. Development of this zone is associated with shoot growth through its functional significance
in supplying water and nutrients (McKee and Morris,
1986). Generally, the xylem zone has a lower content of
total sugars per unit fresh weight than the phloem (Hole,
1996). The central region of the mature carrot taproot,
the pith, is usually not separated from the xylem in
analytical studies (Phan and Hsu, 1973; McKee and
Morris, 1986, Sturm et al., 1995; Zamski and Barnea,
1996).
3 To whom correspondence should be addressed. Fax: +44 1248 370731. E-mail: [email protected]
© Oxford University Press 2000
568 Korolev et al.
Distribution of solutes and dry matter in carrot depends
on variety, size and age of the plant and on the growing
conditions (Hole, 1996). The distribution changes during
taproot development and filling (Platenius, 1934;
Steingröver, 1983; Hole et al., 1987a, b; Steinlein et al.,
1993; Zamski and Barnea, 1996; Shakya and Sturm,
1998). It was proposed that osmotic pressure of the
storage tissues of the carrot taproot is well regulated
during the storage of sugar (Steingröver, 1983; Hole and
McKee, 1988). Concentration of some osmotically active
compounds (potassium, organic acids, reducing and nonreducing sugars) of carrot taproot changes with time,
whereas concentration of magnesium, calcium, amino
acids, and nitrate remain constant (Steingröver, 1983).
Osmotic pressure of the taproot of the carrots grown in
controlled environment (hydroponics and low light
intensity) remained constant from 22–39 d after sowing
(Steingröver, 1983). However, it has been reported for
the taproots of field-grown carrots that there is a 50%
increase in osmotic pressure between 34 and 76 d after
sowing (Hole and McKee, 1988).
Steingröver divided the taproot development into three
distinct periods: first, when no sugars are stored; second,
when reducing sugars are stored and third, when mainly
sucrose is stored (Steingröver, 1983). However, it
remained unclear how those periods related to the taproot
development (e.g. formation of the taproot secondary
structure, Esau, 1940). Moreover, carrot growth before
day 18, well characterized anatomically ( Havis, 1939;
Esau, 1940), remained hidden in terms of solute accumulation and storage.
In physiological studies, the carrot taproot is usually
considered as a bulk storage organ (Platenius, 1934;
Steingröver, 1983; Hole and McKee, 1988). Less frequently the taproot phloem zone was separated from the
xylem and studied independently (Phan and Hsu, 1973;
Sturm et al., 1995). Data on distribution of solutes
between different storage and non-storage tissues of the
taproot are fragmental (Sarkar and Phan, 1974). To our
knowledge there has been no complex study of water and
solute relations at the resolution of tissue and individual
cell of the carrot taproot throughout the period of carrot
growth (from germination to mature plant with the
taproot of marketing size). In this investigation, both the
hand dissection method and SiCSA (single cell sampling
and analysis) were used to obtain tissue samples at fine
resolution. In the case of the cambium only the latter
could be applied.
The following hypotheses will be tested here: (1) that
the accumulation of solutes in the taproot is closely
related to plant growth and taproot development and
that the carrot taproot is an osmotically well-regulated
system and (2) the individual solutes are distributed nonuniformly both between apoplast and symplast of storage
tissue and between the storage and non-storage tissues of
the taproot.
The three growth periods detailed by Steingröver
(Steingröver, 1983) have been revised. (1) From imbibition (day 0) to the formation of the taproot secondary
structure and rupture of the taproot cortex (day 30,
Havis, 1939; Esau, 1940; Hole and McKee, 1988). The
first phase of this period should be emphasized as dark
germination of seeds (0–10 d). This earliest phase corresponds to carrot growth in the field before emergence of
the hypocotyl from the soil. (2) From the end of the first
period until the concentration of hexoses in the storage
tissues of the taproot reaches maximum (day 50, Olden
and Nilsson, 1992; day 60, Ricardo and Sovia, 1974). (3)
From the end of the second period, when the concentration of hexoses starts to decline and sucrose becomes the
major accumulated sugar, until harvest.
In an attempt to estimate the solute distribution
between apoplast and symplast of the carrot taproot both
tissue and single cell sampling and analysis (SiCSA)
( Tomos et al., 1994) were used on the same plants. The
former gave values for both compartments, the latter
only for the symplastic (intracellular) solute
concentrations.
Materials and methods
Growth of plants
Seeds of carrot Daucus carota L. (cv. Bangor) were soaked in
distilled water overnight and germinated in the dark for 10 d in
a roll of filter paper, moistened by either distilled water or Bstrength Long Ashton solution (Hewitt, 1966). Then 15
seedlings were transferred to a trough containing 3.0 l of aerated
B-strength Long Ashton solution and grown hydroponically in
a controlled environment room (25 °C, 15/9 h light/dark regime
with a photon flux density of 1000 mmol photons m−2 s−1). The
nutrient solution was refilled daily and changed weekly.
Preparation of samples
Leaves, petioles, taproot, and fibrous roots were chosen for
analysis. From 30 d on the taproot phloem and xylem zones
split naturally at the cambium when cut. For 30 and 40-d-old
plants the phloem and xylem zones of the taproot were
separated. From day 50 onward, six distinct zone inside the
taproot were chosen and separated using a sharp scalpel. These
were: (1) periderm (0.5–1 mm in thickness) situated on the
surface of the taproot, (2) outer (under the periderm), (3)
middle and (4) inner (close to active cambium) zones of the
phloem region of the taproot, (5) xylem zone (inside the
cambium ring), and (6) pith which is the central region of the
taproot. Note that the separation of phloem from the xylem
zones is straightforward once the cambial ring is complete
(30 d ). The precise separation of the other tissues is much more
difficult and each would have included a small proportion of
the adjacent tissue. It is believed that such contamination has
not influenced the results. 2–3 randomly chosen plants were
used for each analysis and 2–3 tissue samples of 200–300 mg
were sampled per zone per plant. Samples were frozen in sealed
Cell solutes in carrot taproot 569
Eppendorf tubes in liquid nitrogen immediately after separation
and kept frozen at −20 °C until extraction.
Osmotic pressure measurements
Leaves, petioles and the six zones of the taproot were separated
and frozen as described above. After thawing of the samples
and extraction of sap by centrifugation, osmotic pressure was
measured using a Wescor vapour pressure osmometer ( Wescor
Inc. 5100B. Sample volume, 10 ml ).
Measurement of ions
Concentrations of potassium, calcium and sodium in tissue sap
were analysed by flame photometry (PFP 7 flame photometer)
of water extracts. Concentrations of nitrate and malate were
measured enzymatically using nitrate reductase (Beutler et al.,
1986; Anon, 1984) and malate dehydrogenase (Möllering, 1974;
Anon, 1984), respectively.
Measurement of sugars
For the determination of sugars in frozen samples, tissue pieces
(approximately 100 mg) were added to 5 ml of boiling 80%
ethanol and extracted for 2 h. They were then transferred to
60% ethanol for 2 h at 60 °C, and then 4 h in water at 40 °C
(Farrar, 1993). The ethanol extracts were combined, the ethanol
evaporated and the sugars redissolved in deionized water. The
concentrations of sucrose, glucose and fructose were measured
enzymatically using assays linked to the oxidation of glucose6-phosphate (Bergmeyer and Bernt, 1974; Anon, 1984).
Measurement of amino acids
The concentration of individual free amino acids was measured
in the water extracts of entire 10-d-old carrot seedlings. Batches
of seedlings (2 g) were extracted in 50 ml boiling water for
10 min. After cooling, the sample was ground using a mortar,
centrifuged and re-extracted twice with warm water. All extracts
were collected, filtered through Whatman filter paper (No. 1)
on a Büchner funnel and adjusted to standard volume.
Concentration of amino acids was measured in the solution
using HPLC on a Dynamax C18 column ( Webster, 1991). The
results are expressed as the sum total free amino acid
concentration. It was not possible to measure proline using
this column.
Single cell sampling and analysis
Single cell sampling is a development of cell pressure probe in
which a fine glass capillary is used to sample material from
individual cell (Tomos et al., 1994). Single cell sampling and
analysis (SiCSA) are widely used to study water and solutes
relations at the resolution of individual cell (for details see
Tomos and Leigh, 1999).
SiCSA measurements were made on taproot slices to allow
sampling of the cell contents from eight different taproot
regions. The regions were periderm; outer, middle and inner
phloem zones, cambium; outer and middle xylem zone; and
pith. The outer, middle and inner zones (phloem or xylem)
were specified due to the relative position of the cell inside the
taproot rather than by objective physiological assessment. The
cambium was identified under the microscope as several layers
of small closely packed cells, which are situated between phloem
and xylem zones of the taproot.
The taproot slices were immersed in 10 ml of 0.5 mM CaCl
2
solution immediately after cutting to prevent uptake of the
solutes released by damaged cells. Silanazed microcapillaries
filled with silicone oil were used for sampling under the bathing
solution (Tomos et al., 1994; Koroleva et al., 1997). This made
it easier to ensure that no contamination of the sample by
CaCl from the medium occurred during the removal of the
2 from the sampling basin. Three to five cells were
sample
sampled from each taproot region (see above). The samples
were kept in the capillaries between two phases of silicone oil
at −20 °C until analysis of the organic and inorganic solutes.
The concentrations of sucrose, glucose and fructose in the
single-cell samples were analysed by a microfluorometric
enzymatic assay ( Tomos et al., 1994; Koroleva et al., 1997).
Single-cell concentrations of potassium and calcium were
measured by energy dispersive X-ray microanalysis of microdroplets (Tomos et al., 1994, Hinde et al., 1998).
Dry weight determination
For dry weight analysis leaves, petioles, fibrous roots, and two
zones of taproot were separated. These taproot zones were
phloem (periderm, outer, middle, and inner phloem tissues
combined) and xylem (xylem and pith combined). The tissues
were separated, diced and dried in a ventilated oven at 90 °C
for 48 h.
Results
Solute distribution was analysed both in terms of time
and space. The balance of the taproot solutes over the
growth period was described first. Then the spatial distribution of the solutes at two points throughout this period
is described. Together this provides a map of solute
distribution in space and time.
Accumulation of solutes in the taproot during carrot growth
The behaviour of the major solutes of the carrot followed
four phases after seed imbibition. In the first (germination) phase the seedlings maintain their osmotic pressure
using the internal source of solutes. The inorganic solutes
dominated during the second phase. In the third and
fourth these were superseded by hexoses and sucrose,
respectively.
Phase 1 (amino acid accumulation), 0–10 d postimbibition: no difference in osmotic pressure was found
between 10-d-old carrot seedlings germinated in water
and those germinated in B-strength Long Ashton nutrient
solution. Even when seedlings had access to nutrients
during germination in the latter case, the concentration
of inorganic ions in the extracts of the tissues of 10-d-old
carrot seedlings was very low (Fig. 1A, B). This was also
true for malate (Fig. 1C ) and for sucrose ( Fig. 2C ).
Concentrations of glucose ( Fig. 2A) and fructose
( Fig. 2B) were slightly above the 10 mM level. Moreover,
the sum of these solutes, expressed as a predicted osmotic
pressure (66 mosmol kg−1) ( Table 2), was significantly
lower than the directly measured osmotic pressure (225
mosmol kg−1) at this age of the plants ( Table 1). This
was despite the latter being at its lowest point of the
growth cycle. The values of the osmotic pressure (p)
accounted for, and the not-accounted for residual charge
( Table 2), indicate the presence of negatively charged
570 Korolev et al.
Fig. 1. Time-course of potassium (A), nitrate (B) and malate (C )
concentrations in phloem ($) and xylem (&) zones of the carrot
taproot. The ions were measured in seedlings without any separation
(10 d), shoot was separated from root (20 d ), phloem and xylem zones
(together with adjacent periderm and pith) were separated (30–40 d),
xylem and middle phloem zone (free of adjacent tissues) were separated
(50–90 d ).
osmoticum. It was found that the high concentration of
free amino acids (116 mM; not shown) accounted for up
to 50% of the osmotic pressure measured in the extract
of 10-d-old carrot seedlings. Together these identified
solutes (free amino acids, sucrose, glucose, fructose, potassium, calcium, sodium, nitrate, malate, and chloride)
accounted for more than 80% of the directly measured
seedling osmotic pressure.
Phase 2 (ion accumulation): 10–30 d post-imbibition: at
10 d, the seedlings were transferred to a high light intensity
Fig. 2. Time-course of glucose (A), fructose (B) and sucrose (C )
concentrations in phloem ($) and xylem (&) zones of the carrot
taproot. Separation of the shoot, root, xylem and phloem zones of the
taproot as in Fig. 1.
to mimic seedling emergence. The concentration of potassium and nitrate dramatically increased over the next
10 d (Fig. 1A, B, respectively). The maximal values of
individual solutes occurred at day 20 for potassium
(194 mM ) and nitrate (119 mM ), and at day 30 for
malate (26 mM ) (Fig. 1). During this period, the osmotic
pressure increased dramatically. At 30 d after imbibition,
it reached 642 mosmol kg−1 in the leaves, 484
mosmol kg−1 in the petioles and 423 mosmol kg−1
in the phloem and the xylem zones of the taproot
( Table 1). The sap of the fibrous roots retained the value
Cell solutes in carrot taproot 571
Table 1. Osmotic pressure of leaves, petioles, phloem and xylem
zones of the taproot and fibrous roots of the carrot
(mosmol kg−1,±SD, n=4–8)
Time
(d)
10
20
30
60
90
Leaves
Petioles
Phloem
zone
Xylem
zone
Fibrous
roots
301±9***
423±60
427±16
487±3
231±6
159±18
167±22
225±9*
474±7**
642±13
680±20
653±11
484±53
446±25
465±31
423±62
454±34
486±31
*Value for non-separated seedlings, **value for non-separated shoot,
***value for non-separated root. Data for phloem zone at 60 and 90 d
correspond to middle phloem region of the taproot.
Table 2. Osmolality, sum of solute (potassium, calcium, nitrate,
malate, glucose, fructose, and sucrose) concentrations measured
and calculated residual not accounted charge (mequiv l−1) for
phloem and xylem zones of the carrot taproot
Separation of taproot phloem and xylem zone as on Fig. 1.
Time
(d)
10*
30
50–60
90
Taproot
zone
S of solutes
(mosmol kg−1)
p accounted for
(%)
Residual
charge not
accounted for
(mequiv l−1)
Phloem
Xylem
Phloem
Xylem
Phloem
Xylem
66*
340
333
332
319
364
313
29*
80
79
73
75
75
64
19*
102
89
18
30
71
45
*Values for non-separated seedlings.
of osmotic pressure (231±6 mosmol kg−1) close to that
of the seedlings during the germination phase (225
mosmol kg−1) ( Table 1). By this time (day 30), potassium, nitrate and malate could account for up to 80% of
the taproot osmotic pressure ( Table 2). At this point, the
accounted solutes leave a positive charge shortfall of
about 100 meq l−1 indicating that negatively charged
anions, possibly organic other than malate might make
up the difference ( Table 2). Chloride concentration was
less than 10 mM.
Phase 3 (hexose accumulation): 30–50 d post-imbibition:
during this phase the concentration of hexoses increased,
peaked at about 50 d and then decreased ( Fig. 2A, B).
The peak concentration of glucose ( Fig. 2A) was higher
in phloem (154±24 mM ) than that in xylem zone
(108±20 mM ), whereas the concentration of fructose
(Fig. 2B) was less in phloem (58±14 mM ) than in xylem
zone (91±22 mM ).
By 40 d the concentration of nitrate in the xylem and
phloem zones of the taproot had dropped to 1.0±0.9 mM,
whence it stayed constant (Fig. 1B). Potassium and
malate concentrations decreased, more rapidly in the
phloem zone (the minimum were reached at day 40) than
in the xylem zone (minimum at day 50) (Fig. 1A, C ).
Although the concentration of electrolytes (potassium
and malate) dropped during this period, they were not
exported from the tissues and the decrease in the ion
concentration was due to dilution by the accumulated
water. The total amount of potassium, malate and sucrose
in the phloem and xylem tissues of the taproot continued
to rise almost exponentially (calculated from the data in
Figs 1 and 5 and the wet/dry weight ratios, not shown)
due to the increase in their volume.
Phase 4 (sucrose accumulation): 50–90 d post-imbibition:
Concentration (Fig. 2C ) and content of sucrose in the
phloem and xylem zones of the taproot increased more
or less linearly during the entire growth period from 10 d
onward. During phase 4 sucrose became the major accumulated solute and its concentration reached 128 mM in
the middle phloem zone and 151 mM in the xylem zone
of the taproot by day 90 (Fig. 2C ). The osmotic pressure
increased, but non-significantly, in these zones during
phases 3 and 4 (from 30–90 d). Meanwhile it remained
constant in the leaves and petioles and even decreased in
the fibrous roots ( Table 2).
The concentration of potassium in both middle phloem
and xylem zones increased again up to 98 mM and
84 mM, respectively, by day 90 ( Figs 1A, 3A). The
concentration of malate increased in the xylem and in the
inner and outer phloem zones from 50–90 d whereas in
the middle phloem it remained constant ( Figs 1C, 3E).
The concentrations of calcium, sodium and chloride in
the phloem and xylem zones of the taproot were very low
(calcium and chloride <10 mM; sodium <5 mM ) at
all times.
Distribution of solutes in different tissues of the mature
carrot
The distribution of solutes between the various tissues of
the taproot was measured in two different ways; by tissue
extraction and by SiCSA. At 50 d and 90 d postimbibition, using careful dissection with a scalpel, six
distinct tissue zones were excised, weighted and their major
solute concentrations measured (Fig. 3). SiCSA technique
was applied to individual cells within these zones ( Fig. 4).
The finer resolution of the latter technique permitted cells
from two regions within the xylem and from the cambium
zone to be sampled (making eight distinct zones in all ).
The fine resolution, however, is accompanied by cell-tocell variation. In some cases this is due to variation in
planta, in others it is due to experimental error. At 50 d
this variation was large for several solutes (e.g. glucose
and fructose) in some cell types (e.g. xylem zone), whereas
at 90 d the replication was considerably better (Fig. 4).
Single cell potassium was measured in the 90 d plants
only (Fig. 4A), however, no cambium or periderm cells
were sampled for sugars at that time ( Fig. 4B, C, D).
572 Korolev et al.
Fig. 3. Concentrations of potassium (A), sucrose (B), glucose (C ), fructose (D), and malate ( E ) in extracts of different taproot tissues of the 50
and 90-d-old plants. On the x-axis of the figure: periderm (per); outer (ph out), middle (ph mid ), and inner (ph inn) phloem zones; xylem zone
and pith of the taproot are shown.
This might restrict some quantitative conclusions from
the data.
A characteristic pattern for electrolytes was observed
at each time point both for tissue analysis and for SiCSA.
Concentrations of potassium (Figs 3A, 4A) and malate
(Fig. 3E) were lowest in the cambial zone and in the
tissues adjacent to the cambium ring. The concentrations
increased away from these tissues both towards the centre
(pith) and the periphery (periderm) of the taproot. During
the period from 50–90 d there was an increase in concentration of both ions across the taproot radius with the
exception of malate in the middle phloem parenchyma
and a non-significant increase in the pith ( Fig. 3A, E).
Pattern of sugar distribution across the taproot radius
was generally opposite to that of ions (Figs 3, 4), except
for the cambium (see below). At day 50, the radial
distribution of sucrose was symmetrical with the maximum value in the tissue closest to the cambium (inner
phloem zone) and with the minimum in periderm and in
pith (Fig. 3B). In patterns of hexoses at that time, maximum glucose was shifted to the outside (outer phloem
zone) ( Fig. 3C ), whereas that of fructose to the inside
(xylem zone and pith) ( Fig. 3D) of the taproot.
From 50–90 d the concentration of sucrose increased
both in individual cells ( Fig. 4B) and in tissue extracts
( Fig. 3B) from the taproot phloem and xylem zones and
pith with the exception of the inner phloem tissue
( Fig. 3B). At the same time the concentration of glucose
and fructose generally decreased ( Figs 3C, D; 4C, D) or
in several cases remained constant (e.g. fructose in the
phloem tissue, Fig. 3D). The concentration of the free
sugar (sucrose, glucose and fructose) in the cambial cells
of the taproot was below SiCSA detection limit (Fig. 4B,
C, D).
Generally SiCSA measurements agreed with the tissue
extract. In some cases, however, this was not observed.
Cell solutes in carrot taproot 573
Fig. 4. Concentrations of potassium (A), sucrose (B), glucose (C ), and fructose (D) in individual cells of different taproot tissues of 50 and 90 d
plants. On the x-axis periderm (per); outer (ph.out), middle (ph.mid ), inner (ph.inn) phloem zones; cambium (camb); outer (x.out), middle (x.mid)
xylem zones and pith of the taproot are shown.
The tissue extracts ( Fig. 3A) had a higher concentration
of potassium at day 90 than the sap analysed from the
individual cells (Fig. 4A) in all zones of the taproot except
pith. There was also a difference (especially for older
plants) between the tissue and single-cell concentrations
of sugar. At day 90 the concentration of sucrose in
individual cell samples was found to be higher for the
phloem zone of the taproot (some 200 mM; Fig. 4B) than
in whole bulk tissues (some 100 mM; Fig. 3B). This
difference was not observed for xylem zone and pith. In
contrast, the opposite tendency was shown for hexoses.
Concentrations of glucose ( Figs 3C, 4C ) and fructose
(Figs 3D, 4D) were higher in cells than in tissue extracts
of the xylem zone. In this case the values in the phloem
corresponded well.
the total plant dry wt. at 60 d, and up to 50% at 90 d
( Fig. 5B). The dry weight of the xylem zone increased
more slowly and reached 25% of the total plant dry wt.
at day 90. The dry weight of leaves, petioles and fibrous
roots reached their maximum at 70 d and then stayed
constant ( Fig. 5B).
Discussion
In this study a combination of the single-cell (SiCSA)
and tissue sampling and analysis has been used to monitor
the solute accumulation during carrot growth and to map
the radial distribution of the solutes in mature carrot
taproot.
Time-course of solute accumulation
Dry matter accumulation
The dry weight (dry wt.) increase of the plant and its
constituents over the period 33–90 d is shown in Fig. 5.
During this period the dry wt. of the root increased more
rapidly than did that of the shoot (Fig. 5A). The shoot
dry wt. was nearly 35% of the total plant dry wt. at 70 d,
but only 20% at 90 d (Fig. 5A).
The analysis of tissues demonstrated that the dry wt.
of the phloem zone of the taproot accounted for 30% of
This study has generally confirmed the three periods of
solute accumulation in carrot proposed by Steingröver
(Steingröver, 1983). In addition, a dark germination
phase was identified as a period when little solute uptake
from the external medium was detected. Therefore, in
total four phases of solute accumulation during carrot
growth have been identified. Moreover, it is proposed
that the boundaries between the first three can be correlated to clear stages in carrot development.
574 Korolev et al.
Fig. 5. Time-course of dry matter accumulation for the different parts/
tissues of the carrot plant. (A) Whole plant ($), shoot (&) and root
(+). (B) Leaves ($), petioles (&), phloem zone of the taproot (+),
xylem zone of the taproot (,) and fibrous roots (2).
It seems to be that, during the germination phase, the
carrot seedlings absorb water from the external medium,
but use an internal source of solutes. The osmotic pressure
measured in sap of 10-d-old carrot seedlings ( Table 1)
was in a good agreement with the values reported by
Steingröver (Steingröver, 1983). However, the concentration of free amino acids at day 10 was up to 10 times
higher than that measured for mature carrot taproot
(Phan and Hsu, 1973; Nilsson, 1987). The high concentration of amino acids in the young carrot seedlings indicates
the mobilization of storage protein in the seedling tissue
during germination. This phase corresponds to the period
of dark germination of carrot seeds in the field before
appearance of the hypocotyl above the soil. In the greenhouse this period usually lasts for 4–6 d after sowing
(Havis, 1939; Esau, 1940). In this work, the period was
artificially extended till day 10 to get larger seedlings that
were more suitable for transplantation.
The second phase starts with transferring the 10-d-old
seedlings into troughs with aerated nutrient solution. The
only change in the environmental conditions was the large
increase in the light intensity (see growth of plants). The
temperature and the nutrient supply remained unchanged.
During this phase (10–30 d) a remarkable increase in the
sap osmotic pressure was detected ( Table 1). In contrast,
the osmotic pressure of carrot seedlings grown on low,
less physiologically-relevant light levels (260–270 mmol
photons m−2 s−1) remained constant till day 39
(Steingröver, 1983). In these experiments the increase of
osmotic pressure was mainly due to the uptake of potassium ( Fig. 1A) and nitrate ( Fig. 1B) from the nutrient
solution. Accumulation of organic ions such as malate
( Fig. 1C ) kept the osmotic pressure rising even as the
concentration of nitrate fell ( Fig. 1B). However, in terms
of osmotic pressure, the increase in malate ( Fig. 1C ) does
not fully compensate for the nitrate drop (Fig. 1B) and
the final increase in the taproot osmotic pressure
( Table 1). The residual charge balance ( Table 2) indicates
the presence of other anions. The concentration of chloride in tissue sap was very low (<10 mM ). This suggests
that other organic anions (e.g. pyruvate, succinate and/or
oxaloacetate, Phan and Hsu, 1973) might accumulate
along with malate.
At the end of this phase (day 30) the sugar concentration in the taproot (Fig. 2) was at the level of the highest
value reported by Steingröver (Steingröver, 1983).
However, for this experimental system, the sugar (sucrose,
glucose and fructose) accounted for up to 15% of total
osmotic pressure only, whereas the inorganic and organic
ions were the major taproot osmotica at that time. The
anatomical characteristics of this phase are primary
growth of the carrot and development of the taproot
secondary structure (Havis, 1939; Esau, 1940).
It is proposed that the start of the third phase corresponds to the end of the formation of the taproot
secondary structure (30 d, Esau, 1940). The accumulation
of sugar in the taproot tissues starts with a sharp rise in
concentration of hexoses (glucose and fructose), which
increased more than 6-fold during the 20 d and reached
a maximum at day 50 ( Fig. 2A, B). By then, the concentration of inorganic and organic ions had dropped
( Fig. 1). At that time the hexoses accounted for up to
50% of the taproot osmotic pressure. The high concentrations of hexoses in the storage parenchyma were consistent with the high activity of invertase there (Hole, 1996).
The decrease of the hexoses concentration later indicates the beginning of phase 4 (50 d until harvest). The
decline might be a result of the drop of the invertase
activity (Ricardo and Sovia, 1974; Hole, 1996) and was
compensated, in terms of osmotic pressure of the taproot
storage tissue (Table 1), by an increase in the concentra-
Cell solutes in carrot taproot 575
tions of sucrose ( Fig. 2C ), potassium ( Fig. 1A), malate
(Fig. 1C ) and, possibly, pyruvate (Phan and Hsu, 1973).
To summarize, osmotic pressure of the taproot is wellregulated during carrot growth (four phases of solute
accumulation). It would appear that, initially, the demand
for osmotica is satisfied by electrolytes and that these can
be replaced by sugars. Initially, insufficient photoassimilates are available for sucrose accumulation to replace
the electrolytes, so hexoses are used. In this case the same
osmotic pressure can be generated by half the quantity
of sugar. With maturation of the plant, sufficient sucrose
is available to be used to provide the bulk of the osmotic
pressure in much of the tissue.
Spatial distribution of solute
Distribution of solutes along the taproot radius was
mapped at tissue and single cell level. The radial distribution of sugar was generally opposite to that of ions both
at tissue ( Fig. 3) and cell (Fig. 4) level. The highest
concentrations of sugar were detected in the xylem and
phloem parenchymatous storage tissues, whereas those of
ions were measured in the taproot periderm and in the
pith. A notable exception to this is the low concentration
of solutes (potassium and sugars) in the cambial cell
(Fig. 4). Presumably carbon is transported to this tissue
in the form of sucrose and this observation indicates a
high level of the sucrose-cleaving enzymes and rapid
utilization of the hexoses for energetic needs and for
biosynthetic reactions (Sturm et al., 1995). Moreover, the
observation that both sugars and potassium are present
at very low concentrations in the cambial cells raises a
paradox. What are the osmotica of this tissue? It was
shown that the cambial cells do have a significant osmotic
pressure (although considerably lower than that of the
adjacent phloem and xylem), as would be expected for a
tissue that is comprised of cells that must be undergoing
turgor-driven expansion ( Tomos et al., 2000). It can only
be supposed that this nascent tissue resembles that of the
germinating seedling and that it contains significant
quantities of amino acids.
The pattern of the radial distribution of glucose in the
taproot differed from that of fructose at day 50 (Fig. 3C,
D, respectively). The lower concentration of glucose
(Fig. 3C ) in the xylem zone may be due to the preferential
use of the xylem glucose for the cell wall biosynthesis.
The fructose distribution ( Fig. 3D) followed a pattern
opposite to that of glucose. This maintained a constant
concentration of hexoses across the taproot storage tissue.
In both time and space, therefore, it was found that
sugars and electrolytes appear to act as alternative solutes,
with osmotic pressure being largely maintained. A similar
situation has been found in sugarbeet tissue where sugars
and non-sugar solutes have a similar reciprocal relationship in their concentration (Milford, 1973), the ratio
being dependent on cell size. In the case of carrots it is
clear that the pattern is developmental. This may also be
the case in sugarbeet, where the low sucrose concentration
is found in the oldest cells. These developmental profiles
of tissue potassium are in contrast to those of primary
root development, where the small dividing cells of meristematic tissue have the highest potassium concentration
due to a higher proportion of cytoplasm in the tissue
(Jeschke and Stelter, 1976). Potassium concentrations are
thought to fall as the proportion of vacuole in the tissue
rises. In maize roots, however, reciprocal radial gradients
of electrolytes and sugars have been found when SiCSA
was used to measure radial profiles of solutes across a
fully expanded region near the root tip (Pritchard et al.,
1996).
The authors had expected to be able to estimate the
solute distribution between the apoplast and the symplast
of the mature carrot taproot by comparing the solute
concentrations measured in the same plants at both tissue
( Fig. 3) and at individual cell (Fig. 4) level. It was
expected that the latter would be a precise value for
individual protoplasts, while the former would be a
mixture of inter- and extracellular solution (cf. Leigh and
Tomos, 1983). In some cases, the apoplast appeared to
dilute the bulk tissue extract, while in others it had the
reverse effect.
The concentration of potassium in the tissue slices was
higher compared with that in the cells from the same
zone of the taproot (Figs 3A, 4A). It is suggested that
part of the explanation for this is that potassium is
accumulated in the apoplast of the taproot tissues.
Accumulation of sugars in cells leads to an increase in
the cell osmotic pressure and, as a result, in potential rise
of the cell turgor. Apoplasmic potassium, in this case,
might act as an external osmoticum to counteract this
effect and maintain (osmotically adjust) the turgor in the
storage cell. A similar situation was found for storage
tissue of red and sugar beet roots (Leigh and Tomos,
1983; Bell and Leigh, 1996), where it is believed to be a
part of the mechanism of avoiding a damagingly high
turgor. However, the magnitude of the changes was much
larger than can be explained in this way. For example,
concentration of sucrose in the cells of phloem parenchyma (Fig. 4B) was nearly twice as high as that in the
phloem tissue slices (Fig. 3B). This would require a
volume of low sucrose solution equal to that of the
parenchyma protoplasts to be present. In addition, this
pool of solution would have to dilute any influence of
the sucrose contained in phloem sieve elements. These
elements presumably contain high sucrose concentrations,
but their small volume (Esau, 1940) means that the
amount of sucrose they contain is small. It seems unlikely
that this can only be due to a dilute apoplast solution. It
is possible that the tissue contains a population of lowsucrose cells that were not observed in the SiCSA analysis.
At the same time as noted above the concentration of
potassium in the tissue slices ( Fig. 3A) was twice as high
576 Korolev et al.
as that in the cells ( Fig. 4A). This is consistent with the
unidentified low-sucrose compartment containing potassium at a concentration much higher than the parenchyma
cells analysed by SiCSA. The concentration of hexoses in
the phloem tissue slices (Fig. 3C, D) was similar to that
in the cells ( Fig. 4C, D).
In the xylem zone of the taproot the sucrose behaviour
was different. The concentration of sucrose in the parenchyma cells ( Fig. 4B) was similar to that in the slices of
the xylem zone ( Fig. 3B). This was also unexpected, as it
was envisaged that the contents of the xylem vessels
would dilute the bulk tissue concentration below that of
the parenchyma symplast. Therefore, a high sucrose concentration in the apoplast of the xylem zone could be
expected.
Finally, it is proposed that the storage task is partially
separated between different taproot tissues. Taproot periderm acts as a protective layer. Damage to this tissue
usually causes taproot splitting (McGarry, 1995). It is
also a relatively poor source of nutrients for invading
pathogens and grazing feeders (cf. the epidermis in cereal
leaves, Fricke et al., 1994; Koroleva et al., 1997). Phloem
and xylem parenchyma accumulate sugars in the form of
hexoses and sucrose (Hole, 1996); with some discrimination towards glucose in the phloem and fructose in the
xylem. The pith of the mature taproot is the preferred
place for the storage of water and ions.
Acknowledgements
Dr GS Webster and Dr JS Chung are gratefully acknowledged
for their help with the analysis of free amino acids. This work
was financially supported by BBSRC research grant to RB,
ADT and JFF.
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