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in the name of god

Ion distribution measured by electron probe X-ray microanalysis
in apoplastic and symplastic pathways in root cells in sunflower
plants grown in saline medium
REZA EBRAHIMI
1,* and SC BHATLA
2
1Department of Soil Science, University of Guilan, Rasht, Iran
2Department of Botany, Delhi University, Delhi, India
*Corresponding author (Fax, +0098-131-6690281; Email, rj_ebrahimi@yahoo.com)
Little is known about how salinity affects ions distribution in root apoplast and symplast. Using x-ray microanalysis,
ions distribution and the relative contribution of apoplastic and symplastic pathways for delivery of ions to root xylem
were studied in sunflower plants exposed to moderate salinity (EC=6). Cortical cells provided a considerably
extended Na+ and Cl− storage facility. Their contents are greater in cytoplasm (root symplast) as compared to those
in intercellular spaces (root apoplast). Hence, in this level of salinity, salt damage in sunflower is not dehydration due
to extracellular accumulation of sodium and chloride ions, as suggested in the Oertli hypothesis. On the other hand,
reduction in calcium content due to salinity in intercellular space is less than reduction in the cytoplasm of cortical
cells. It seems that sodium inhibits the radial movement of calcium in symplastic pathway more than in the apoplastic
pathway. The cell wall seems to have an important role in providing calcium for the apoplastic pathway.
Redistribution of calcium from the cell wall to intercellular space is because of its tendency towards xylem through
the apoplastic pathway. This might be a strategy to enhance loading of calcium to xylem elements and to reduce
calcium deficiency in young leaves under salinity. This phenomenon may be able to increase salt tolerance in
sunflower plants. Supplemental calcium has been found to be effective in reducing radial transport of Na+ across
the root cells and their loading into the xylem, but not sodium absorption. Supplemental calcium enhanced Ca2+
uptake and influx into roots and transport to stele.
[Ebrahimi R and Bhatla SC 2012 Ion distribution measured by electron probe X-ray microanalysis in apoplastic and symplastic pathways in root
cells in sunflower plants grown in saline medium. J. Biosci. 37 713–721] DOI 10.1007/s12038-012-9246-y
1. Introduction
Salt stress is one of the major abiotic stresses limiting crop
growth and productivity. NaCl salinity can cause injury to
plants due to absorption of toxic levels of sodium and
chloride ions by roots and their transport to shoots (Taiz
and Zeiger 2006). It can also antagonize the uptake of other
essential nutrients, such as nitrogen, phosphorus, potassium
and calcium, thereby inducing their deficiency or imbalance
in plant organs or cells in spite of their abundance in soil or
the growth medium (Tester and Davenport 2003; Wahome
2003). Deleterious effects of NaCl salinity on plant growth
and mineral nutrition are, thus, attributed to a decrease in
osmotic potential of root growth medium, specific ion
toxicity, nutrient imbalance and deficiency as a result of
disruption of ion uptake (Kwon et al. 2009).
In low salinity, osmotic solutes in plant organs are normally
not in sufficient concentration to have major ability for
inward water movement in such situations. Some of plants
try to accumulate high levels of compatible solutes in plant
organs in order to create a water potential gradient for inward
water movement (Ebrahimi 2010). Under NaCl salinity, the
enhanced rates of Na+ and Cl– accumulation in roots and its
transport to shoots also alters the activity of various apoplastic
enzymes in the constituent cells of plant roots (Philippar
and Soll 2007).
Sunflower cultivars vary from being very sensitive to semitolerant
of salt stress (Ashraf and Tufail 1995; Francois 1996).
http://www.ias.ac.in/jbiosci J. Biosci. 37(4), September 2012, 713–721, * Indian Academy of Sciences 713
Keywords. Apoplastic pathway; calcium; root; sunflower; symplastic pathway
Published online: 13 August 2012
Sunflower crop grows well on a wide range of soils, such as
sandy loam, black and alluvial soil. The ideal soil pH for
growth of sunflower is from 6.5 to 8. An understanding of
the factors affecting the growth of sunflower plants in
degraded lands, such as saline soils, is necessary in order
to increase the yield of oilseeds and maintain good oil
quality. NaCl salinity (50 and 100 mM) has been reported
to affect seedling survival rather than seed germination in
sunflower (Delgado and Sanchez-Raya 2007). In addition,
sodium and chloride ions generally accumulate more in the
vegetative parts as compared to reproductive parts in
sunflower plants (Ebrahimi and Bhatla 2011). The differences
in their ion concentrations is due to the fact that vegetative
parts are exposed to salinity for much longer periods and the
reproductive parts being fed by phloem are better protected
as well as exposed for relatively shorter durations.
Roots directly suffer from soil salinity. Differences in ion
distribution in different subcellular compartments in various cell
types in roots may explain differences in plant response to soil
salinity. In saline soil conditions, roots must continue to acquire
essential nutrients while excluding toxic ions (Davenport 2007).
In order to achieve this, roots try to preferentially take up
essential ions, such as K+ and Ca2+, over toxic ions, such as
Na+ and Cl−, in saline conditions in the growth medium.
Enhanced Na+ exclusion by plant roots is an important trait
that increases salt tolerance in many crops growing in saline
soils (Yeo 2007). In some crops raised in saline soils, enhanced
Cl– exclusion from crops roots is associated with
increased salt tolerance rather than Na+ exclusion (Storey
and Walker 1999). Not much is known about the putative
mechanisms that determine Cl– exclusion except that it may
involve restriction of Cl– uptake and its increased sequestration
in root vacuoles (Storey et al. 2003).
Na+ may enter xylem stream through many pathways
(Tester and Davenport 2003). Some of those pathways have
been identified at the molecular and electrophysiological
levels (Storey et al. 2003). They include different K+ carriers
and non-selective cation channels (Tyerman and Skerrett
1999). In some plants, pathways that bypass membrane
transport processes may also be involved in the translocation
of Na+ into the transpiration stream and its translocation to
the shoots (Shabala 2007). Cl– uptake into the cells is facilitated
by H+/Cl– symporters (Storey et al. 2003). At high
salinity, Cl– might also enter the root cells passively through
anion channels, although at low salinity, this is reported to be
unlikely (Mengel and Kirkby 2001).
With this background information in view, ion contents within
the individual root cells from specific regions have been
determined by electron probe x-ray microanalysis using frozen
specimens. The x-ray microanalysis was restricted to the regions
of the primary roots. Thus, the work was primarily confined to a
region of the root that accumulates ions from the external medium.
Localization of elements in individual root cells or
subcellular compartments of plant tissues using x-ray analytical
electron microscopy has been successfully applied earlier in a
number of investigations (Drew et al. 1990; Kosegarten and
Koyro 2001; Patrick 2001; Conn, and Gilliham 2010).
On the basis of published data on the organ level in sunflower,
40 mM NaCl in Hoagland solution (EC=6 dS/m) is a
critical content of salinity that can cause visible injury in
sunflower leaves (Ebrahimi and Bhatla 2011).
Lower concentration does not causes significant impacts
on sunflower growth, and at higher salinity levels sunflower
plant cannot survive. In order to understand how sunflower
roots control Na+ and Cl– transport from root to shoot and
prefer to accumulate these ions in root cells, a comparative
study has been undertaken in the present work with the
following aims:
1. Determination of Na+, K+, Ca2+ and Cl− contents in
epidermis, outer and inner cortical cells and stele
2. Determination of how 40 mM NaCl in the growth medium
alters cellular ion distribution in primary root
3. Determining whether there are differences in Na+, K+,
Ca2+ and Cl– contents in the cytoplasm and apoplast in
the cells of primary roots. For this comparative analysis,
various cells from roots were analysed for differences in
ion accumulation
4. Investigation of whether the ratios ofK+/Na+ and Ca2+/Na+
in the cytoplasm differ from those in the apoplast
5. Finally, Examining the effect of 10 mM supplemental
calcium on Na+, K+, Ca2+ and Cl− loading into xylem
and ratios of K+/Na+ and Ca2+/Na+ in root cells under
NaCl salinity
2. Materials and methods
2.1 Plant material and seed germination
Seeds of sunflower (Helianthus annuus L. cv.Morden)
were obtained from National Seeds Corporation, India.
Uniform-sized seeds were selected and surface-sterilized
with 0.1% mercuric chloride for 5 min, thoroughly washed
with deionized water and soaked for 12 h in glass distilled
water. Seeds were germinated in plastic pots filled with
fine silica sand (< 2 mm) and were uniformly irrigated once
daily. Emergence of radicle was taken as a sign of germination.
The formation and extension of hypocotyls was observed after
48 h. When the cotyledons had fully expanded, three uniform
seedlings were retained for growth in each pot.
Potted plants were maintained in a controlled environment
room and subjected to 16 h photoperiod diurnally at
25±1°C and 80±5% relative humidity. Throughout the
experiment, plants were irrigated daily and uniformly
with half-strength Hoagland nutrient solution (pH=6)
714 Reza Ebrahimi and SC Bhatla
J. Biosci. 37(4), September 2012
containing 40 mM NaCl with or without supplemental
calcium. Control plants were not subjected to NaCl
treatment.
Plants were harvested after 30 days of growth and their
roots were washed with distilled water. Thirty-day-old plants
were cut to separate roots. Experiments were conducted in
three independent replicates per treatment, each replicate
consisting of three plants.
2.2 Sample preparation for x-ray microanalysis of ions
Samples were processed according to Zhao et al. (2005).
Briefly, root segments of 5 mm×5 mm were cut from near
the root tip from 30-day-old plants. The tissue samples were
put into small cages of aluminium gauze and plunged into
liquid N2–cooled iso-pentane and propane (v/v 1/3) for rapid
freezing and freeze-dried at −80°C in the lyophilizing chamber.
Modified T-shaped valves were used for infiltration with
diethyl ether under vacuum at 25°C for 24 h after freezedrying.
The samples were then cut into small pieces for
infiltration with styrene-methacrylate.
They were then transferred into gelatine capsules and
polymerized at 60°C for 7 days. The embedded materials
were cut using a LKB Nova ultramicrotome to obtain
70–90 nm thick samples, using a diamond knife. The
sections were pasted on the stub and coated with gold
for observation by transmission electron microscope,
with emphasis on cortical cells in roots. The energy
dispersive x-ray microanalytical studies were carried
out under standardized conditions, as described in
Bucking and Heyser (2000), using a Philips 420 electron
microscope fitted with PV9100 EDAX system.
Element distribution was documented as a peak-tobackground
ratio (P:B) in order to diminish the effects of
surface irregularities of the sections during analysis. Due to
standardization problems with x-ray microanalysis, P:B ratio
was used as a semi-quantitative measurement of the element
levels in different cellular compartments. X-ray microanalysis
of ion contents in roots was also done by field emission
scanning electron microscope (FESEM) coupled with
EDAX (Vesk et al. 2000).
The sections were examined by HITACHI–H 800
Field Emission Scanning Electron Microscope fitted with
EDAX- 910 energy disperses x-ray micro analyser. The
accelerating voltage was 12 kV with a takeoff angle of
25°. The counting time for all analysis was 60 s (spectra
were collected for 60 s) and the data were expressed as
counts per second (cps) of an element peak after subtraction
of the background. Six measurements per section
were carried out for each tissue compartment.
Specimen drift was minimized by keeping the specimen/
stage temperature at a constant –145°C.
3. Results
The validity of ion content measurements in subcellular compartments
depends more on the preparative procedure more
than on themicroanalysis itself. The aimof cryopreservation in
this work was to freeze root samples rapidly so that damage is
not caused by the formation of ice crystals. As a preparative
technique to fix solutes in their natural compartments, rapid
freezing is more versatile than chemical precipitation for both
scanning and transmission electron microscopy (Flowers
2007). There was a minimum gap (<15 s) between cutting
and freezing in this work. Once frozen, there would be no
opportunity for diffusion to take place during sub-sampling
of small pieces of roots used for freeze substitution. In this
work, a scanning beam of 13×14 μm was found suitable for
probing the sections (inner contents of cells). This technique
provided useful information about ion distribution in apoplastic
and symplastic pathway in salinized and nonsalinized
root cells in sunflower plants. Through this method,
clear distinctions were made from cell to cell in roots.
3.1 Anatomy of root and its changes
After freezing the root sections in liquid nitrogen, structural
preservation of the root cells was found to be satisfactory.
Freezing in iso-pentane+propane and rapid transfer into liquid
N2 gave improved results. Epidermis of roots is characterized
by the absence of cuticle and stomata. Cortical cells are
considerably homogenous in shape and well developed.
Cortex is a major component of the ground tissue in sunflower
root and is composed of several layers of thin-walled, loosely
arranged parenchymatous cells with intercellular spaces.
3.2 Energy spectra
Differences in ionic contents of root cells in control and
NaCl-treated plants were revealed by analysing energy spectra
on the basis of peak area and P:B ratio of elements as a
percentage of the mass fraction analysed. In each EDAX
spectrum of elements, the y-axis showed counts per second
(cps) and x-axis showed energy in kilovolts. Energy spectra
of sodium, potassium, calcium and chloride ions in root
cells, measured by x-ray technique, showed significant differences
in response to salinity although some relocation of
diffusible elements, such as Na+ and K+, during the embedding
procedure cannot be completely excluded. Data are
expressed as counts per second (cps) of an element peak
after subtraction of the background. Six measurements per
section were carried out for each compartment. X-ray counts
were typically in the range from 0.11 to 11.82 cps and the
dwell time was 60 s. Peak emissions for carbon and oxygen
were also detected but have not been discussed in this study.
Ion distribution in apoplastic and symplastic pathways in root cells 715
J. Biosci. 37(4), September 2012
Representative energy dispersive x-ray spectra for the cytoplasm,
intercellular space and cell wall of cortical cells
demonstrated clear peaks for K+ and Ca2+ under non-saline
conditions (figure 1A) or for Na+ and Cl− in saline conditions
(figure 1B). Na+ peak in the cytoplasm of outer cortical
cells was in the range of detection limit (Na+=0.11) in nonsaline
conditions. In some cases, sodium content was not
detectable after focusing the electron excitation beam onto
the cytoplasm of epidermal cell in this condition. Figure 1
also shows the energy spectra of the characteristic x-ray
analysis of ions present in the cell wall of cortical cells in
sunflower plant roots after growing in non-salinemedium. In this
region, sodium was detectable (=0.65 cps). Na+ and Cl− enrichment
in cortical cells in saline condition, as compared to
control, was confirmed by the relative percentage of Na+ and
Cl− in comparison with the distribution of K+ and Ca2+.
Furthermore, the peak for potassium ions was clearly larger than
that for calcium in the cytoplasm, cell wall and intercellular
space of cortical cells in non-saline conditions (figure 1A).
The x-ray measurement analysis of ions in roots
grown in saline medium revealed a distinct Na+ signal
in root cells and it could be detected in the cytoplasm, cell
wall and in intercellular space. The peak area for chloride
was generally bigger than that of sodium in saline conditions
(figure 1B).
3.3 Profile of Na+ and Cl−
In sunflower plants grown in non-saline conditions, content
of sodium ions in root cells was very low and that of chloride
ions a little higher than sodium. In the roots of control plants,
distribution of Na+ and Cl− followed a different pattern. The
highest mean contents of sodium and chloride ions were in
the cytoplasm of inner and outer cortical cells, respectively,
and lowest contents were observed in the cytoplasm of
epidermal cells for both of ions.
Under 40 mM NaCl salinity, chloride content in root cells
was more than of other ions, and sodium content was more
than potassium and calcium. In this condition, the highest
mean content for sodium and chloride ions was in the cytoplasm
in cortical cells and lowest for Na+ in xylem elements
and for Cl− in epidermal cells. In terms of absolute quantities,
most of the Na+ and Cl− were in the vacuoles that
occupied a large volume of the cell cytoplasm in saline
conditions.
In non-saline conditions, Na+ content in roots was mainly
detectable in the cytoplasm of inner cortical cells, whereas
the levels in the cytoplasm of outer cortical cells were near
the x-ray microanalytical limit of detection. In a few cases,
Na+ was not detectable in the cytoplasm of epidermal and
outermost cortical cells. Sodium content increased slightly
A B C
.
A
K
+
Na
+
Cl
-
Elemental content (counts per seconed)
Energy (K.eV)
Epidermis Epidermis Epidermis
Ca2+
Outer cortex Outer cortex Outer cortex
Inner cortex Inner cortex Inner cortex
Intercellular space Intercellular space Intercellular space
Figure 1. X-ray spectra of the element content (counts per second) in root cells (A: Half-strength Hoagland Solution (HHS), B : HHS +
40 mM NaCl, C: HHS + 40 mM NaCl + 10 mM CaSO4).
716 Reza Ebrahimi and SC Bhatla
J. Biosci. 37(4), September 2012
from the outermost to innermost cortical cells in non-saline
conditions. It was lowest in the cytoplasm of epidermal cells,
higher in the intercellular space and highest in the cytoplasm
of innermost cortical cells in roots grown under non-saline
conditions.
The mean content of Na+ in root cells increased due to
40 mM NaCl salinity. Under saline conditions, Na+ content
in all root cells was always more than that in non-saline
conditions. Accumulation of these ions in the symplastic
pathway in cortical cells was always more than that in the
apoplastic pathway. The increase in sodium and chloride
ions contents in root cells appeared to be larger in the
presence of 2 mM calcium (control) as compared to
10 mM calcium in the saline growth medium. Sodium content
in the cytoplasm of the inner cortical cells was higher
than that in the cytoplasm of epidermal cells. Sodium content
in the intercellular space was less than that in the cytoplasm
in cortical cells in presence of NaCl salinity. Sodium content
in root cells under saline conditions was significantly different
with 2 or 10 mM calcium treatments. It decreased in
salinized root cells in the presence of 10 mM calcium as
compared to 2 mM calcium in the growth medium. The
maximum and minimum reduction in sodium content in root
cells due to 10 mM supplemental calcium was observed in
xylem and epidermal cells, respectively.
Under non-saline conditions, chloride content in the cytoplasm
of cortical cells was higher than that in the cytoplasm
of epidermal cells. Ch#loride content in the cytoplasm
of outer cortical cells and in cell wall was almost similar to
that in control root cells (figure 2). It was lower in intercellular
spaces in comparison with the cytoplasm of inner
cortical cells under non-saline conditions. It was least in
the cytoplasm of epidermal cells. NaCl salinity increased
chloride content in all root cells significantly. Maximum
increase in chloride content was observed in the epidermal
cells due to NaCl salinity (tenfold increase as compared to
that in controls). However, chloride content in epidermal
cells was still less than in other cells under saline conditions.
Chloride content was the highest in the cytoplasm of inner
cortical cells among root cells grown under 40 mM NaCl
salinity (sixfold increase as compared to that in controls).
Chloride content decreased due to 10 mM supplemental
calcium sulphate in root cells in the presence of 40 mM
NaCl. The minimum and maximum reduction in chloride
content was observed in the cytoplasm of outer cortical cells
and in xylem elements due to supplemental calcium sulphate,
respectively.
3.4 Profile of K+ and Ca2+
The content of potassium and calcium ions was considerably
higher than that of sodium and chloride ions in root cells in
non-saline conditions. In general, among cations, potassium
content in root cells was more than others in sunflower
plants grown under non-saline conditions. The highest mean
content of K+ was in the cytoplasm of cortical cells and
lowest in the cytoplasm of epidermal cells (figure 2). In roots
grown under non-saline conditions, K+ content in the cytoplasm
of cortical cells was twofold higher than that in the
intercellular space of these cells. In non-saline conditions,
the potassium gradient in root cells was much steeper than
that in salinized root cells. In non-salinized roots, the chemical
gradient of potassium decreased substantially across the
cortex and this gradient was probably steeper than the potassium
gradient across the salinized roots.
NaCl salinity (40 mM) induced a lowering of K+ content
in the apoplast and symplast of cortical cells in roots. In salttreated
plants, the profiles of K+ and Na+ showed a significant
decrease in potassium and an increase in sodium content
in root cells. Minimum potassium content was detected in
cell wall under NaCl salinity. It seems that NaCl salinity
decreases both the absolute amount of K+ in root and the
ratio between that transmitted to the stele and that retained
by the root cells themselves. However, potassium content
was almost constant across the root cells under salinity
treatment (figure 2). The salt-induced reduction in K+ content
in root cells was larger in the presence of 2 mM calcium
(control) than 10 mM calcium in the growth medium
(figure 2).
0
1
2
3
4
Sodiuum (cps)
0
2
4
6
8
epidermal
cells
outer
cortex
inner
cortex
xylem
elements
free space
Ca++/Na+ ratio
0
4
8
12
16
20
epidermal
cells
outer
cortex
inner
cortex
xylem
elements
free space
K+/Na+ ratio
0.2
0.6
1
1.4
1.8
epidermal
cells
outer
cortex
inner
cortex
xylem
elements
free space
Ca lcium (cps)
0
2
4
6
8
10
Potassium (cps)
0
1
2
3
4
5
Chloride (cps)
Figure 2. Ions content and ratio in different root cells in sunflower plants irrigated with Hoagland solution ( ) or plus 40 mM NaCl ( )
or plus 40 mM NaCl + 10 mM CaSO4 ( ) for 30 days in sand culture medium.
Ion distribution in apoplastic and symplastic pathways in root cells 717
J. Biosci. 37(4), September 2012
Supplemental calcium improved K+ content in cortical
cells and xylem elements in sunflower roots in saline conditions.
It was significantly higher in the xylem elements in
roots subjected to 10 mM calcium, in comparison with
similar cells from controls. Potassium content in free space
of cortical cells was similar with and without supplemental
calcium in saline medium (figure 2).
Calcium content was considerably less than potassium
content in all root cells in sunflower plants under nonsaline
conditions. Calcium content in the cytoplasm of outer
cortical cells was more than that in the cytoplasm of inner
cortical cells (figure 2). Under non-saline conditions, calcium
content in the cytoplasm of cortical cells was more than
that in the intercellular space of cortical cells. The highest
Ca2+ content was detected in the cell wall of cortical cells.
Among the cell wall, cytoplasm and intercellular space of
cortical cells, the minimum content of calcium was observed
in intercellular space in non-saline conditions.
In the presence of 40 mM NaCl in Hoagland solution
(with 2 mM Ca2+), the mean Ca2+ content in root cells
decreased as compared with that in roots of control plants.
In roots exposed to salinity, the symplastic and apoplastic
distribution of Ca2+ was almost uniform but the relative
reduction of calcium due to salinity in the symplastic pathway
was more than in the apoplastic pathway. A markedly
higher calcium level was detected in the outer cortical cells
of roots treated with 10 mM of calcium as compared to roots
grown in presence of 2 mM calcium treatment in saline
medium (figure 2).
3.5 Profile of K+/ Na+ and Ca2+/Na+
The K+/ Na+ ratio in the cytoplasm of cortical cells of roots
decreased from the outer cortex to the inner cortex under
non-saline conditions (figure 2). This ratio was similar in the
epidermal cells and cytoplasm of outer cortical cells and was
threefold greater than that of the inner cortex under nonsaline
conditions. In the presence of 40 mM NaCl in the
growth medium, K+/Na+ ratio was almost uniform in all root
cells. NaCl salinity (40 mM) significantly reduced this ratio
in root cells as compared to non-saline conditions. Maximum
reduction was observed in outer cortical cells. It decreased
100-fold as compared to that in non-saline conditions.
Supplemental calcium improved this ratio slightly. K+/Na+
ratio was slightly higher in 10 mM calcium in comparison
with 2 mM calcium in all root cells in saline conditions. The
minimum and maximum improvement was observed in epidermal
cells and xylem elements, respectively, but the improvement
was not significant in any of these cells.
The Ca2+/Na+ ratio was, however, lesser than the K+/Na+
ratio in all root cells in non-saline and saline conditions. This
ratio decreased from outer to inner cortical cells in sunflower
roots grown under non-saline conditions (figure 2). There
were significant differences in Ca2+/Na+ ratio in root cells in
control plants. It was maximum in epidermal cells and minimum
in intercellular space of cortical cells in roots grown
under non-saline conditions. NaCl salinity decreased this
ratio in all root cells as compared to control. In the presence
of salinity this ratio was similar in all cells in root. The
maximum reduction was observed in epidermal and outermost
cortical cells. Supplemental calcium improved this ratio
slightly in all root cells, and in the cell wall it was more than
others.
4. Discussion
4.1 Ion distribution in apoplastic and symplastic pathways
in root cells is dependent on their long-distance transport
and roles in specific zones
Calcium is taken up by plant roots either by the apoplastic or
the symplastic pathway (White 2001). The relative contribution
of these two pathways for delivery of calcium to the
xylem in different plants is not known (Cholewa 2000). The
results obtained in this work showed that salinity affects both
pathways in sunflower roots. The content of Ca2+ in the
cytoplasm is normally very low and is regulated by the
activity of membrane-bound transporters. Intracellular level
of calcium must be maintained as low as 0.1–0.6 μM within
the cytosol in order to avoid phosphate precipitation (Mengel
and Kirkby 2001).
Ion distribution within the root cells is dependent on their
long-distance transport to the shoot and their biochemical/
osmotic roles (Storey et al. 2003). There is a variation in the
distribution of Na+, K+ and Ca2+ in the sunflower root cells
which could be correlated with their spatial position within
the root. These results suggest that various ions may adopt
different uptake pathways in roots.Whereas K+, Cl− and Ca2+
are predominant elements in the cytoplasm of root cells
under non-saline conditions, Na+ is predominant under
NaCl salinity in root cells. Under saline conditions, Na+
and Cl− contents are greater in the cytoplasm of cortical cells
as compared to intercellular spaces of cortical cells. For plant
roots grown under non-saline conditions, a similar analysis
reveals that the cytoplasm of root cells contain very little
sodium because for growth and development of sunflower,
K+, Ca2+ and Cl− are recognized as essential nutrients while
the role of Na+ remains debatable (Rajendra 2007). It seems
that sodium ions in low concentration can substitute for
potassium in raising cell turgor in sunflower (Ebrahimi and
Bhatla 2011). Most plants growing in normal soils contain
very little sodium in root cells (Mengel and Kirkby 2001).
Chloride content in cells of sunflower roots is generally higher
than sodium. Their concentrations are, however, equal in salty
irrigation water in sand culture medium. Uptake rate of
chloride ions by root cells in sunflower is higher than that of
718 Reza Ebrahimi and SC Bhatla
J. Biosci. 37(4), September 2012
sodium ions (Ebrahimi and Bhatla 2011), as also reported
earlier in some other plants (Mengel and Kirkby 2001).
The noteworthy observation in the present work is that the
total ion content in the cytoplasm is greater than that in the
intercellular space of cortical cells. This forms strong evidence
that an important factor in salt damage in sunflower plants is
not dehydration due to extracellular accumulation of sodium
and chloride ions, as also suggested in the Oertli hypothesis
(Oertli 1968; Flowers et al. 1991). Previous work has shown
differential Na+ accumulation in sunflower leaves as a property
of roots (Quintero et al. 2007 and 2008). In the region of
roots analysed in this work (cortex), it was observed that Na+
content mirrored the Na+ content in shoots in low salinity but
not in high salinity. It seems that maximum chloride ions get
accumulated in cytoplasm (in vacuoles) of cortical cells, as
reported by other workers as well (Storey et al. 2003).
4.2 Salinity reduces potassium content in the cytoplasm
of cortical cells more than in the intercellular space
Potassium is involved in many metabolic processes in plant
cells. In the cell cytoplasm, K+ is involved in the neutralization
of soluble and macromolecular anions, and it contributes
to the osmotic potential of plant cells (Mengel and Kirkby
2001). A disorder in potassium nutrition in root cells has
been observed to be one of the nutritional problems induced
by NaCl salinity in sunflower (present work). There is a
strong competition between sodium and potassium ions for
uptake by root hairs and plant cells. Both are monovalent
cations and have slight differences in their ionic radii.
Therefore, Na+ can easily substitute for K+ in less specific
processes, such as raising cell turgor. Under saline conditions,
the cell wall seems to have an important role in
providing potassium for cell cytoplasm in roots, and potassium
from the apoplastic pathway gets redistributed to the
symplastic pathway because the radial transport of K+ in
plant roots is symplastic (Drew et al. 1990).
4.3 Cell wall provides calcium for other cell
compartments in roots under salinity
Calcium is an important constituent of the middle lamella
and cell walls, preventing membrane damage and leakage,
strengthening wall structure and increasing the cohesion of
cell walls (Fageria 2009). It also plays an important role in
the regulation of growth and developmental processes and
acts as an intracellular signal molecule. Current investigations
reveal that in sunflower roots, calcium content of the
cell wall is considerably reduced by NaCl salinity. The mean
cytoplasmic Ca2+ content in root cells of sunflower plants
grown in the presence of 40 mM NaCl is still sufficient to
meet the aforementioned biochemical requirements, but the
cell wall–associated calcium in cortical cells of roots grown
in similar saline conditions is not physiologically sufficient
(Flowers et al. 1991). Displacement of cell wall–associated
calcium by sodium and magnesium ions is likely to increase
membrane permeability and loss of Ca2+/Na+ selectivity.
Reduction in calcium content in the free space of cortical
cells due to salinity is less than that in their cytoplasm. This
might be a strategy to enhance loading of calcium to xylem
elements and to reduce calcium deficiency in young leaves
under saline conditions because calcium usually reaches the
xylem through the apoplastic pathway in plants (Mengel and
Kirkby 2001). It seems that sodium ions inhibit the radial
movement of calcium from external solution to xylem elements
in sunflower roots.
4.4 Supplemental calcium seems to control sodium
loading into xylem but not sodium absorption by root
from growth medium
Some beneficial role of supplemental calcium on improvement
of calcium content in the cell wall during NaCl salinity
has been observed in sunflower plants, as also reported
earlier for some other plants (Kwon et al. 2009).
Supplemental calcium seems to control sodium loading into
the xylem. This appears to be the key for young sunflower
plants to tolerate NaCl salinity. Leaves show NaCl toxicity
symptoms earlier than root. That is why roots try to accumulate
Na+ and Cl−, instead of loading them into the xylem
for transport to the shoot. In the present work, supplemental
calcium (in the form of CaSO4) has been found to be effective
in reducing the radial transport of both Na+ and Cl− in
sunflower roots and long-distance transport to the xylem.
This phenomenon thereby reduces leaf injury in sunflower.
Sodium content in epidermal cell cytoplasm in roots grown
in saline conditions with 2 or 10 mM calcium was almost
similar. It seems that supplemental calcium in saline medium
cannot reduce the sodium uptake by roots. Supplemental
calcium seems to reduce sodium loading to xylem elements
but not sodium absorption by roots. More studies are, however,
required to explore the exact mechanism of supplemental
Ca2+ in reducing Na+ transport and loading to the xylem
and enhancing Ca2+ uptake and influx to roots in sunflower.
Apoplastic sodium gradient may play a role in the K+/Na+
gradient across the cortical cells in sunflower roots. The K+/
Na+ ratio increases in cortical cell and xylem elements due to
supplemental calcium sulphate in saline conditions. Zaman
et al. (2002) reported that K+/Na+ ratio improved due to
sulphur application in sunflower.
4.5 Role of roots in salinity tolerance and nutrient balance
Sunflower is a semi-tolerant plant to NaCl salinity as a result
of accumulation of Na+ and Cl− in roots as the main osmotic
regulators. Under moderate salinity (EC=6 dS/m), about
Ion distribution in apoplastic and symplastic pathways in root cells 719
J. Biosci. 37(4), September 2012
65% of Na+ and Cl− are restricted in sunflower roots
(Ebrahimi and Bhatla 2011). The effect of 10 mM supplemental
Ca2+ treatment is significant in reducing Na+ transport
across the root cells because of an enhancement of Ca2+
uptake and Ca2+ influx into roots, transport of Ca2+ to stele
and alleviation of growth inhibition in roots caused by salinity
in sunflower ‘cv. Morden’, but a little alleviation of calcium
content in cell wall. The cell wall seems to provide calcium for
other regions of the cell in roots under moderate salinity in
sunflower. Extracellular accumulation of Na+ and Cl− is not
the primary factor for salinity damage in sunflower roots. An
increase in solute concentration in root cells is sufficient to
balance the salinity of the external medium and is evidence
for osmotic adjustment of the cells in sunflower roots.
Generally, ion uptake by roots shows marked selectivity
for potassium over sodium, but since this is of limited value
in excluding sodium or maintaining adequate potassium content
in the root cytoplasm, additional regulatory mechanisms
are required to minimize this problem in sunflower root.
Cortical cells in sunflower roots act as accumulators of Na+
and Cl−. Subcellular distribution of these ions under saline
conditions mainly occurs in the cytoplasm of inner cortical
cells, with vacuoles having the highest content. As a consequence
of large number and volume of cortical cells, they
could provide a considerably extended Na+ and Cl− storage
facility in sunflower roots. In this context, it is interesting to
note that the mean ion concentrations in roots, as derived
from x-ray microanalysis of the cortical cells, is comparable
to that obtained by flame photometry or atomic absorption of
whole-root samples (Ebrahimi and Bhatla 2011).
This would seem to indicate retention of Na+ and Cl− in
the cortex, with transmission of K+ to the stele, from where it
can be transported to the shoots. On the basis of maximum
reduction in potassium and calcium content in the cell wall
due to salinity, it seems that the cell wall of cortical cells has
an important role in saline conditions to provide potassium
and calcium for the cytoplasm. This phenomenon may be
able to increase salt tolerance in sunflower roots exposed to
salinity. Na+ and Cl− absorbed by root hairs are partly translocated
to the stele and transferred to the shoots, which can
cause an inhibition of metabolic processes in the leaves in
sunflower plants grown in saline soils.
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MS received 27 November 2011; accepted 17 July 2012
Corresponding editor: RENU KHANNA-CHOPRA

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