Download Ultrastructural and physiological changes in root cells of Sorghum

Journal of Experimental Botany, Vol. 48, No. 308, pp. 693-706, March 1997
Journal of
Experimental
Botany
Ultrastructural and physiological changes in root cells of
Sorghum plants [Sorghum bicolor x S. sudanensis cv.
Sweet Sioux) induced by NaCI
Hans-Werner Koyro1
Botanisches Institut der Tiera'rztlichen Hochschule Hannover, BOnteweg 176, D-30559 Hannover, Germany
Received 20 December 1995; Accepted 12 September 1996
Abstract
The xerophytic, but salt-sensitive Sorghum cultivar
'Sweet Sioux' is known as an ion excluder with a high
K/Na selectivity at the plasmalemma and tonoplast of
epidermal root cells. The aim of this study is the correlation of salt-effected changes in physiological parameters with structural and ultrastructural changes in
root cells. The investigation was carried out with root
cells because these cells are most directly exposed to
the growth medium.
Sorghum bicolor xS. sudanensis cv. Sweet Sioux
plants were grown under steady-state conditions on
nutrient solutions with or without 40 mol m " 3 NaCI.
Sorghum sustained this treatment but showed several
salt-induced structural and physiological changes
which were studied in various cell types of the root tip.
(1) NaCI salinity led to a shorter growth region and
to salt-induced alterations in the chemical and
physical properties of the cell walls in the root tips.
(2) Salt treatment also increased the membrane surface in root cells: root cells showed an increase in the
quantity of vesicles in the epidermis and in the middle
cortex cells. Additionally, some of the epidermis cells
of salt-treated plants revealed a characteristic build-up
of transfer cells, suggesting an increase in membrane
surfaces to increase the uptake and storage of
substances.
(3) The number of mitochondria increased in the
epidermal and in the cortex cells after salt stress thus
indicating an additional supply of energy for osmotic
adaptation and for selective uptake and transport
processes.
(4) In the epidermal cytoplasm NaCI stress led to a
significant decrease of the P, K, Ca, and S concentra1
tions accompanied by an increase of Na concentration.
Electron micrographs show an increase in electron
optical contrast within the cytosol and in the matrix of
the mitochondria. These results are discussed with
regard to the possibility of influence on the part of
metabolic functions.
(5) The NaCI concentrations were seen to increase
and the K concentrations to decrease during salt
stress in the vacuoles of the epidermis and cortex
cells. The salt-induced increase in vacuolar NaCI concentrations of epidermis and cortex cells are in the
region 2 cm behind the root tip, which is sufficient for
an osmotic balance towards the growth medium.
Additional solutes are necessary 0.5 mm behind the
root tip to facilitate osmotic adaptation.
The results show ultrastructural changes caused by
an Na-avoiding mechanism characterized by a high
level of energy consumption. The exclusion of Na from
the symplast seems to lead additionally to a decrease
in cytoplasmic concentrations of such essential
elements as Mg, P, S, and Ca and is thus responsible
directly (via energy supply in mitochondria, homeostasis, selectivity of K over Na) or indirectly (via enzyme
conformation, cytoplasmic hydration) for the ultrastructural degradation indicated. The salinity-induced
multiplicity of structural and functional changes within
cell compartments constitutes a group of indicators
for the limited NaCI tolerance of Sorghum.
Key words: Sorghum bicolor x S. sudanensis,
structure, salt tolerance, NaCI, Ca-deficiency.
ultra-
Introduction
Sorghum plants are cultivated mainly in sub-humid and
semi-arid climate regions (Corlett et al., 1994; Gorz et al.,
Present address: Zentrale Biotechnische Betriebseinheit im Strahlenzentrum der Justus-Liebig-Universitat Giessen, D-35392 Giessen, Germany.
Fax: +49 641 993 0099. E-mail: Hans-Werner.Koyro©strz.uni_giessen.de
© Oxford University Press 1997
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Koyro
1987; Hodson and Evans, 1995). Osmotic adaptation to
drought results from the accumulation of organic compounds (for example, sucrose, glucose and fructose) in
the vacuoles and with proline or glycinebetaine as an
osmoprotectant, in the cytoplasm (Basnayake et al, 1995;
Bhaskaran et al, 1985; Jones, 1978; Weimberg et al.,
1984). The metabolic costs of drought-induced osmotic
stress seem to be compensated for by a proportionally
high reduction in biomass synthesis (Auge et al., 1995;
McCree, 1986; Osmond, 1980).
In contrast to these results, vanadate-sensitive ATPase
activity in Sorghum roots increases 5-fold under salt stress
(40 mol m~3 NaCl) while the rate of ATPase activity in
nitrate- and azide-sensitive tissue is six times higher
(Koyro et al., 1993). It is not clear whether the energy
requirement induced by salt treatment can be compensated for by growth reduction.
Much effort has been invested in the conjunctive use
of saline irrigation waters in semi-arid regions to grow
Sorghum plants (Sharma et al., 1994). However, Sorghum
is resistant to low water potential in the soil caused by
drought, but highly sensitive to NaCl salinity (Amzallag,
1994; Weimberg et al., 1984; Yamane, 1993). This suggests
a direct effect of Na + or Cl~ on the metabolism. The
root tip acts as a finely-tuned sensor for different kinds
of stress (Colmer et al., 1994; Roth and Bergmann, 1988).
The outer cortex and especially the epidermis cells have
a key function in the uptake of minerals into the symplast
of the plant. Direct exposure of the root cortex led us to
expect structural changes in these cells, which may explain
the low salt tolerance of the Sorghum species.
At the tissue level, roots show a broad spectrum of
structural adaptations to environmental changes. NaCl
salinity or Fe deficiency can, for example, lead to reduced
growth and to a shorter elongation zone (Bernstein et al.,
1993; Wilson and Robards, 1978; Clarkson et al., 1987).
A diminished O2 supply can induce development of an
aerenchyma (Stelzer and LSuchli, 1977).
A considerable amount of data is available on saltinduced changes at the cellular level. Comparative investigations have been conducted with drought-sensitive
Sorghum and salt-tolerant Spartina plants grown under
steady-state conditions with or without 40 mol m~3 NaCl
(Koyro et al., 1993). Salt treatment caused an increase
of epidermal IMP (intramembraneous particle) frequencies in the plasmalemma and tonoplast. This is interpreted
by the authors to mean an increase in membrane- bound
enzymes (such as ATPases). This supposition was confirmed by a salt-induced increase in ATPase activity (F-,
T- and P-ATPase) per ng protein.
In Suaeda maritima and A triplex hastata the root
endodermis develops faster and the Casparian strip was
significantly bigger after the addition of NaCl to the
nutrient solution (Kramer et al, 1978; Hajibagheri et al,
1985). Moreover, Nishizawa and Mori (1984) have dem-
onstrated the presence of vesicular (pinocytotic) ion
transport from the plasmalemma to the tonoplast.
There can also be differences in the behaviour of single
cells within the same tissue with regard to environmental
stress. Several authors have detected transfer cells in
solitary epidermal or hypodermal cells (Kramer et al,
1977, 1978; Gunning, 1977) and Fricke et al. (1994) has
demonstrated a correlation between position and ion
composition of single leaf epidermal cells in barley. Huang
and Van Steveninck (1990) have shown that salinity
causes many plastids in cortical root cells to adopt varying
amoeboid shapes, but have also determined variations in
the density of the cytoplasm.
Functional differentiation is usually accompanied by
differences in (ultra) structure. The salt-induced increase
in the activity of all three ATPase types in Sorghum root
tips and, simultaneously, changes in the IMP-frequencies
in the tonoplast and plasmalemma of epidermis cells
constitute only one example of such an interrelationship
(Koyro et al, 1993).
Many analytical studies have been carried out on the
correlation between the mineral content of Sorghum
plants and attendant specific soil conditions (Richardson
and McCree, 1985; Press and Stewart, 1987). Studies of
element distribution at the cellular level are, by comparison, rare. The aim of the present paper is to describe
salt-induced changes in cell structures and cytoplasmic
and vacuolar element concentrations in epidermis and
cortex cells of Sorghum roots in order to identify the
physiological reason for sensitivity to NaCl salinity and
to pinpoint those metabolic processes in the plant where
genes for salt tolerance might be expressed (Munns,
1993).
Materials and methods
Plant growth
Seeds of Sorghum bicolor x S. sudanensis (L.) Moench cv. Sweet
Sioux were washed for several minutes with warm tap water
and imbibed for 1 d in the dark (25 °C) in an aerated 0.2 mol
m~ 3 CaSO 4 solution. After this, the seeds were then plotted on
plastic grids and germinated for 1 d in large plastic containers
(10 1) in a 0.2 mol m~ 3 CaSO 4 solution. Subsequently, the
seedlings were cultivated under photoperiodic conditions (16 h
light/8 h dark) in a growth cabinet (Koyro et al, 1993). After
1 week, the plants were placed in a nutrient solution with 1 mol
m " 3 K + (Epstein, 1972). Three weeks later, one batch of plants
received 40 mol m~ 3 NaCl (added within 2 d to the growth
medium). The remainder of the culture served as a control. The
nutrient solutions were renewed weekly.
Light microscopy (Lm)
The light-microscopical investigation was performed with a
Zeiss photomicroscope III equipped with a Normarsky lens.
The photomicroscope's Hg-HP lamp (HBO 50 W) suffices to
induce fluorescence. Fresh material was sectioned by hand with
a razor blade 1-2 cm behind the root apex (n = 20). Sections
were stained by mounting them in aniline blue (Hughes and
Ultrastrvctural and physiological changes in Sorghum
Gunning, 1980). A Zeiss UG exciter filter and Zeiss 41 barrier
filter (passed A >410nm) were used to detect the fluorescence
induced by aniline blue (specifically for callose). Background
autofluorescence was substracted photographically from the
aniline blue-induced fluorescence.
A sequence (>10 sections) of 1-10 fim thin cross-sections
was cut in fixed, dehydrated and resin embedded root tips in
order to investigate the influence of NaCl on the cell
development of root tissues, thus enabling morphometric
analysis of equivalent sections from each treatment (n = 32
cells). Root tips were therefore fixed for 4 h with 1%
glutaraldehyde in 50mol m~ 3 cacodylate buffer (pH 7.2). To
imitate the osmotic differences of the nutrient solutions, 40 mol
m~ 3 NaCl was added to the fixative of salt-treated plants. After
washing the root tips three times in buffer (with and without
NaCl) the tips were post-fixed for 4 h with a 1% OsO 4 solution
in buffer. After a few washings in distilled water, the root tips
were dehydrated in acetone (Sitte, 1962). They were then
transferred stepwise from acetone to propylenoxid (25%, 50%,
75%, 100% propylenoxid), and replaced at the end of this
procedure by resin (Technovit, Kulzer). Embedded material
was sectioned with a glass knife on an LKB ultramicrotome
(Ultrotome I). The sections were stained with methylene blue
(in 2% ethanol) on a heating plate (60 °C).
Transmission electron microscopy (TEM)
Root tips were prefixed with 1% glutaraldehyde in 50 mol m" 3
cacodylate buffer (pH 7.2). 40 mol m~ 3 NaCl was added to the
fixation solution of salt-treated plants. The washing of the
roots, post-fixation with Os0 4 , and dehydration were performed
in the same way as for light microscopical examination. After
this, roots were embedded in ERL^1206 resin (Spurr, 1969),
and polymerized at 70 °C for 8 h. Ultrathin cross-sections
(30-70 nm) were cut 0.5-1 mm behind the root apex with the
Ultrotome I (LKB) or Ultracut E (Reichert-Jung) ultramicrotome. The sections were stained for 8 min with 1% uranyl
acetate and for 4 min with 5% lead citrate before they were
observed in a transmission electron microscope (Zeiss EM 10A
or Zeiss EM 902). The number of mitochondria, vesicles and
plastids per cell was calculated in more than 30 cells per
treatment (10 sections per cell) from three cultures.
Electron microscopy on freeze-fractured root tips
Root tips (n= 16 per treatment) were placed in Balzers specimen
carriers and frozen within 515 ms under high pressure
(> 210 MPa) in a Balzers HPM 010. Freezing the specimen by
rapid immersion, with a 2 m s~l entry velocity of the specimen
into liquid propane (Reichert K.F 80) was employed as an
alternative procedure (Kaeser et ai, 1989). The frozen root
tips were fractured longitudinally, etched for 1 min (2.4 x
10" 4 Pa; 163 K), shadowed with platinum-carbon (1 nm Pt/C
layer) at 45°, and, stabilized with carbon at 90° (25 nm C layer,
Balzers BA 360 M). The specimens were subsequently thawed,
rinsed with water, cleaned for 72 h with chromosulphuric acid
and for 24 h with sodium hypochlorite. Afterwards, the replicas
were washed several times with distilled water, mounted on
pioloform coated copper grids, and viewed in a transmission
Zeiss EM 10a electron microscope. Micrographs were taken
0.5-1 mm behind the root tip.
X-ray microanalyses (EDXA)
Root tips (15 tips per treatment) were mounted on a
Cu-specimen stub together with droplets of standard solutions
(40, 80, 120 mol m " 3 K + , Na + , Cl", 20, 40, 80 mol m " 3 Ca 2 + ,
Mg2+, PO 3 ~, SOJ". The roots were cut transversely with a
695
blade cooled with liquid nitrogen on a cryo-stage under high
vacuum conditions, then transferred to the column of an ETEC
Autoscan SEM equipped with a KEVEX Si/Li detector for
X-ray count harvesting. The electron beam was focused 0.5 mm
behind the root tip on cytoplasm and vacuoles of epidermal
cells and on vacuoles of middle cortex cells, and 2 cm behind
the root tip on vacuoles of both cell types (15 sections per
treatment and stage).
Subsequent procedure of the quantification have been
described elsewhere (Koyro, 1989; Stelzer et al, 1988).
Results
Figure 1 shows a typical cross-section of controls and
NaCl-treated roots (« = 32). Intercellulars are in the process of development between all cortex cells of root
controls 400 ^m behind the root tip. Actively dividing
regions are visible in the endodermal and epidermal cell
layers. In all cortex cells, with the exception of the
endodermis and epidermis cells, a multitude of small
vacuoles is present. In this region the typical elements of
the stele are visible, showing only some vesicles in the
metaxylem element vessels and in the sieve-tube.
Advanced development of nearly all tissues is one consequence of the salinization: the middle cortex cells show
a large central vacuole; the endodermal and epidermal
cells have large vesicles, the metaxylem vessel element no
longer contains living contents.
The reduced degree of enlargement in cells adapted to
40 mol m~3 NaCl was not the result of absorption of
lignin and suberin into the cell walls. Observations made
with solutions of Sudan III or Phloroglucinol/HCl did
not show any of these substances at 1 or 2 cm from the
root tip (results not shown). However, in contrast to the
control roots, an additional deposition of callose into the
cell walls of the outer cortex cells and of the endodermis
was generally observed after salt treatment (Fig. 2a, b).
Changes in wall structure can influence the elongation
of the cell. The observations made by transmission electron microscopy with freeze-fracture replicas of epidermal
cells show differences in the wall in response to salinity
treatment (Fig. 3). At 1 mm behind the root tip of the
controls, thin microfibrils were arranged mainly (in 87%
of the cells investigated) in a parallel array. In corresponding cells of the salt-treated roots, the microfibrils almost
exclusively (91% of the investigated cells) formed a closely
meshed network.
Comparison of the ultrastructures in conventional fixated and embedded samples was undertaken between 0.75
and 1 mm behind the root tip. The observations showed
conspicuous differences between the control and salinity
treatment. In the epidermis and cortex cells of control
roots the stroma of the plastids, the matrix of the mitochondria, the nucleoplasm and the cytoplasm all appear
to have a comparable high density (Fig. 4a, b). No
uniform picture was presented by salt-treated epidermis
696 Koyro
Fig. 1. Light micrographs (Normarsky optics) of cross-sections 400 pm from the junction of the meristem and root cap. There are conspicuous
differences in the state of development of root tissues of the (a) control and (b) NaCl-treated plants (bar, 100 ^m); r: epidermis; c: cortex; ic:
intercellulars; ed: endodermis; ph: phloem; mx: metaxylem vessel element.
Ultrastructural and physiological changes in Sorghum
697
Fig. 2. Cross-sections of Sorghum roots 1-2 cm behind the root tip, stained with aniline blue, (a) Aniline blue-induced fluorescence can be seen in
the stele, but not in the cortex of controls, (b) The aniline blue positive deposits show up clearly in the outer cortex cells of NaCl-treated plants; r:
epidermis; c: cortex; ed: endodermis.
698
Koyro
Fig. 3. Electron micrographs of freeze-fracture replicas from epidermal cells of Sorghum root tips, (a) In control roots, the cellulose microfibrils are
arranged mainly in a parallel array (bar; 100 nm). (b) In corresponding cells of the salt-treated roots, the microfibrils form a close-meshed network.
Ultrastructural and physiological changes in Sorghum
699
r
Fig. 4. Electron micrographs showing epidermis cells of controls (a, b) and salt-treated roots (c, d). (a, c) The survey of the epidermis cells
demonstrates the identical stage of development, (b) The cytoplasm and the enclosed organells appear to be very dense. The plastids frequently
store large amounts of starch, (d) Differences of density in the cytoplasm are visible between neighbouring epidermal cells. In cells where the
cytoplasm appears to be less dense, the chnstae of the mitochondria are swollen (arrow), p: plastid; me: mitochondnum; the bars represent 10 fim.
cells as a whole. Some cells showed no change at all in
comparison with the controls (right cell, Fig. 4c, d). In
many corresponding cells of salt-treated roots, the cytoplasm, the matrices of the mitochondria and the nucleo-
plasm contained a lower concentration of heavy metal
binding substances, which points to a decreased density
in their matrices (left cell in Fig. 4c, d). The sole exception
was the stroma of the plastids.
700
Koyro
The walls of the epidermis cells are very thin, and
differences in the ultrastructure of resin embedded samples
cannot be observed after NaCl treatment. Nevertheless,
in some epidermal cells of the salt-treated roots, invaginations are apparent in the plasmalemma and cell wall,
giving the overall appearance of a transfer cell (Fig. 5).
However, it is also possible, that the structure shown is
the root cell's reaction to bacterial infection. These protuberances in the cell wall are observed only in cells with a
high cytoplasmic density.
The influence of salinity on the number of mitochondria, small vesicles and plastids in epidermal and middle
cortex cells is listed in Table 1. NaCl treatment increases
the amount of vesicles in the epidermis and in the middle
cortex cells. The amount of mitochondria increased significantly only in the epidermis. The number of plastids did
not change significantly in any of the recorded cells.
The epidermis cells develop more slowly than the
cortical cells in the root tip (Fig. 6, see also Fig. 1). An
Table 1. Average number of mitochondria, vesicles and plastids
in epidermal (with the exception of transfer cells) and cortical
root cells of controls and NaCl-treated Sorghum plants: all
values are given as mean ± SD
Epidermis
Mitochondria
Vesicles
Plastids
Cortex (middle)
Mitochondria
Vesicles
Plastids
Control
+ 40
NaCl
12.1 ±0.7
2.1 ±0.5
2.8 ±0.4
15.8 ± 1.8
4.2±1 8
2.8±0.2
13 0 ± 1.4
0.5 + 0.2
l.8±1.2
14.3 ±0.1
3.2±0.5
1.9±0.3
accurate EDX measurement of the cytoplasm was only
possible in epidermal cells 0.5 mm behind the root tip
(Fig. 6). K and P are the dominant elements in the
cytoplasm and vacuoles of epidermal and cortex cells
(Fig. 7). P and K concentrations are always higher in the
Fig. 5. Electron micrograph showing the interface between a transfer cell and a 'normal' epidermal cell from a salt-treated Sorghum root. The
christae of the mitochondria are only swollen in cells where the cytoplasms appear to be less dense (arrow).The enrichment of mitochondna (me),
endoplasmic reticulum and ribosomes next to the wall is characteristic of this cell type. The transfer cell shows the typical invaginations of the
inner cell wall (bar. 1 ^m).
Ultrastructural and physiological changes in Sorghum
701
Fig. 6. Cryo-scanning electron micrograph from transverse fracture-faces of Sorghum root tips 0.5 mm behind the tip (bar, 10 fim); c: cytoplasm,
N: nuclei, v: vacuole.
vacuoles of the epidermis cells, as they are in the cortex
cells. Salinity leads to a small increase in Na and a
significant decrease in P, K, S, and Ca in the cytoplasm
(Fig. 7a). Apart from an increase in NaCl and a decrease
in Ca, the vacuole shows no significant salt-induced
changes in element concentrations (Fig. 7b).
The composition of the vacuolar solutes is similar in
the epidermis and cortex cells (Fig. 7b, d). It is obvious
that salinity produces a greater increase in epidermal
NaCl than in the cortical vacuoles 0.5 mm behind the
root tip. K concentrations decrease after salt treatment
only in cortex cells of this region.
P concentrations decrease and Cl concentrations
increase during cell development in epidermal and cortical
vacuoles (Fig, 7c, e). Salinity causes a significant decrease
in K and Ca concentrations in epidermal and cortex cells
2 cm behind the root tip. Only in this region are saltinduced increases in Na and Cl sufficient for osmotic
balance towards the growth medium.
Discussion
Salt and drought stress was associated in root tips of
Sorghum with a shortening of the growth region. The
elongation zone of Sorghum leaves was also shorter in
salinity despite the fact that the duration of elongation
growth associated with a cell during its displacement from
the leaf base was longer in salinized leaves than in the
control (Bernstein et ai, 1993). Additionally, transpiration, growth and photosynthesis were strongly reduced
by salinity (Auge et ai, 1995; Koyro, 1989; Nagy et ai,
1995). It has been shown that the turgor, the yield
threshold and the wall extensibility have an influence on
the cell growth (Cosgrove, 1981). As reported for other
root cells (Pritchard et al., 1991) Sorghum roots exhibit
complete regulation of single-cell turgor when osmotically
stressed with 40 mol m " 3 NaCl (Koyro, 1989). This
result suggests an effect of NaCl in Sorghum on the yield
threshold and/or on the wall extensibility. The present
paper shows several salt-induced changes in the cell wall
of outer cortex cells from Sorghum which could explain
a change in wall rigidity.
(1) Salinity induces the storage of the j3-l,3-D-glucan,
callose, in the cell walls of the outer cortex cells in
Sorghum.
(2) Freeze etching replicas present a typical secondary
cell wall in epidermis cells of controls whereas
salt-treated cells present mainly primary cell walls.
702 Koyro
20
10
0
80
70
(c)
60
50
40
30
20
10
0
Na
Mg
Cl
Ca
Na
Cl
Ca
3
Fig. 7. Cytoplasmic (a) and vacuolar (b-e) element concentrations (mol m ) in epidermal (a-c) and cortical cells (d, e) of Sorghum roots grown
under steady-state conditions on nutrient solutions without D or with • 40 mol m" 3 NaCl. Mean values and SD (vertical bars). The measurements
were taken 0.5 mm (a, b and d) or 2 cm (c and e) behind the root tip.
Binzel et ai, (1987) have shown that alterations in the
physical properties of the cell wall take place in cells
adapted to NaCl stress. Carbon is partitioned for osmotic
adjustment at the expense of cell wall synthesis. There is
considerable data supporting the hypothesis that the
hydrolysis of glucans (i.e. xyloglucan) permits wall expansion (Cleland, 1981; Lipetz, 1970). Callose, 0-1,3-Dglucan, is an insoluble polysaccharide found in many
plant tissues; it seems however, to be particularly associated with senescence, dormancy, and injury. Numerous
small deposits on the wall between the pits are followed
by the deposition of a continuous layer on the inner wall
surface (Currier, 1957). It is possible that the deposition
of callose hinders the expansion of the root cells.
The arrangement of microfibrils in a parallel array
(Fig. 3) in the longitudinal cell walls supports the enlargement of cells in one (i.e. longitudinal) direction (Lflttge
et ai, 1989). In corresponding cells of salt-treated roots,
cells show no preference for cell enlargement. The
arrangement of microfibrils in the longitudinal cell walls
in a closly meshed network supports the uniform cell
extension.
Apart from indicating changes in the quantity of organelles, it was possible to demonstrate a salt-induced
increase in electron optical-contrast in the cytosol and in
the matrix of the mitochondria, but not in the stroma of
the plastids. Similar results have been shown for Zea
mays and barley (Huang and Van Steveninck, 1990, Nir
et al., 1966; Siew and Klein, 1968).
It is difficult to interpret results of samples fixed with
Ultrastructural and physiological changes in Sorghum
glutaraldehyde and osmium, dehydrated in acetone and
embedded in resin because of the possible presence of
artefacts. It should, nevertheless, be possible to attribute
salt-induced differences between structures directly or
indirectly to physiological changes. Increase in contrast
suggests an imbibed cytosol and matrix. It is believed
that there is a connection between the decrease in Mg, P,
S, K, and Ca concentrations in the cytoplasm, as shown
for bulk frozen samples (Fig. 7), and the decrease in the
electron optical contrast (Fig. 4). Dissolved ions and
especially mineral nutrients such as Ca, Mg and S, alter
the physical properties of the solvent water by forming
hydration shells around the ion, and also alter the properties of the protein molecule through interactions
(Marschner, 1993). Disulphide bonds as well as ionic or
covalent bonds with Mg and Ca are essential components
in the hydration, stability, and conformation of many
enzymes and in hydration of the cytoplasm. A decrease
in the concentration of these elements could, therefore,
be responsible for an imbibition of the proteins into the
cytoplasm as suggested for resin embedded Sorghum root
cells. Despite many investigations of the effects of NaCl
on enzyme activty (Jennings, 1976), the results indicate
that it is not ion excess of NaCl but ion deficiency (Ca,
Mg, P, K) that is a possible reason for the decrease in
enzyme activity in the cytoplasm. Further experiments
are needed to investigate enzyme activities under more
natural conditions.
Hydration of the cytoplasm may also be changed by
an acidification. An increase in the Na + /K + -exchange at
the tonoplast has been put forward as one possible reason
for a change of the pH in the cytoplasm (Garbarino and
Dupont, 1989; Uribe and Luttge, 1984). Contrary to this
theoretical possibility, NaCl induces a reduction of the
pH gradient across the tonoplast in Sorghum bicolor root
tips by vacuolar alkalization rather than cytoplasmic
acidification (Colmer et al., 1994). An increase in pH
degrades the roots and lowers the rate of Na and Cl
exclusion in excised roots exposed to NaCl stress
(Tsuchiya et al., 1995).
Ca seems to be beneficial in maintaining a high
pH-gradient between cytoplasm and vacuole and sustains
the putative driving force for N a + transport from the
cytoplasm into the vacuole via a N a + / H + channel as well
as levels of cytoplasmic K + , cytoplasmic and vacuolar
phosphate (Colmer et al., 1994). The decrease in Ca in
cytoplasm and vacuoles of epidermis cells correlates with
inhibition of membrane functions such as K + / N a + selectivity (Colmer et al., 1994). Ca can alleviate the inhibitory
effect of NaCl on root growth by maintaining plasmamembrane selectivity of K over Na (Zhong and Lauchli,
1994). Salt-induced changes of the ultrastructure and
element composition in epidermal root cells can be
explained in terms of a Ca deficiency leading to loss of
control over the cytoplasmic homeostasis. Further salinity
703
experiments are needed with varying Ca supply to investigate the sites of action of Ca.
Salinity increased the amount of vesicles in the epidermal and in the middle cortex cells of Sorghum roots.
It could not be established whether these vesicles were of
tonoplast or cytoplasmic origin. Advance of vacuolation
in the apical region of barley root apices could be effected
by treatment with 50 mol m~ 3 NaCl (Huang and Van
Steveninck, 1990). Water stress led to an increase of small
vesicles (< 1 /xm) in root cells of Zea mays (Nishizawa
and Mori, 1981) as did Ca-deficiency in potato sprouts
(Hecht-Buchholz, 1979). The build-up of many small
vesicles led to an increase in membrane surface. The
authors interpret this increase in the number of vesicles
as a limited enhancement of the transport capacity and
storing activity of the afflicted cell and as a possible
means of excluding excess ions from the cytoplasm. The
ability of plants to control the uptake and compartmentation of Na and Cl constitutes a mechanism for salt
tolerance (Flowers and LSuchli, 1983). A multiplicity of
small vesicles processes a larger surface than one big
vesicle, hence a higher exchange capacity (Na versus K ) .
This system enables a plant cell to avoid ion toxicities,
imbalances or interactions between substances in the
cytoplasm.
Additionally, the build-up of transfer cells was only in
single epidermal cells of salt-treated plants, and never in
hypodermal cells. Transfer cells have been shown to be
present for several species in epidermal or hypodermal
cells (Kramer et al., 1978; Kramer, 1983; Winter, 1988).
It has been claimed that this is an indirect effect due to
salt-induced iron deficiency (Kramer et al., 1980).
There appears to be a correlation between the buildup of transfer cells and an extensive exchange of substances at the barrier between apoplast and symplast
(Gunning, 1977; Hill and Hill, 1976; Thomson et al.,
1988). With regard to NaCl tolerance, transfer cells have
been considered to be connected with K + / N a + discrimination (Kramer, 1983) with increases in the exchange
capacity between compartments. The advantage of an
increase in vesicular and transfer cell (plasmalemma)
surface indicates a higher and more selective uptake and
storage of substances.
The decrease in electron density within the matrix of
the mitochondria seems to reflect a degeneration of the
mitochondria and therefore a decreasing efficiency. Opik
(1965, 1966), Schwab et al. (1969) describe similar
changes for mitochondria. In root tips of a salt-sensitive
cultivar of Agrostis stolonifera (grown on 100 mol m~ 3
NaCl) the sacculi of the mitochondria were swollen and
their number per organelle was lower than in controls,
whereas there was no salt effect on the ultrastructure of
mitochondria in a salt-tolerant cultivar (Smith et al.,
1982). Visible changes in the sacculi (Fig. 5) and ER also
point to a Ca-deficiency (Fig. 7; Koyro and Stelzer, 1988)
704 Koyro
and may also be an indication of increased respiratory
activity. Ca is often discussed in connection with
membrane integrity, cell wall composition and salt tolerance (Greenway and Munns, 1980; Lynch and Lauchli,
1988). The structural changes observed in mitochondria
within epidermal cells could therefore be caused by a
Ca-deficiency in the matrix.
The number of mitochondria was higher in the epidermal and cortical cells of Sorghum plants treated with
NaCl than in the controls. K-deficiency and NaCl salinity
in salt-sensitive species can also lead to an increase in the
number of microbodies as well as to an increase in the
number of mitochondria (Hecht-Buchholz et al., 1971;
Hecht-Buchholz, 1983; Walker and Taiz, 1988). The saltinduced increase in F-ATPase activity in Sorghum roots
corresponds to the increase in the number of mitochondria
in the cortex cells and, therefore, suggests an additional
supply of energy for osmotic adaptation and for selective
uptake and transport processes (Koyro et al., 1993).
Salinity does not change the amount of plastids in all
three cell types and, therefore, has no influence on the
interpretation of changes in the F-ATPase activity. Koyro
and coworkers discuss the high energy requirement of
salt-stressed plants, explaining it as the consequence of
the very selective uptake of K over Na (SKNa) and of
osmotic adaptation to organic compounds. Especially
0.5 mm behind the root tip (Fig. 7a-c) organic compounds seem to be necessary for the osmotic adaptation
in the cytoplasm and in the vacuoles of root cells.
The salt-induced increase in the number of mitochondria and the swollen christae, when taken together with
the increase of the F-ATPase activity (Koyro et al., 1993)
as documented, points to increased availability of
metabolic energy or to lower metabolic activity per
mitochondrion. Sorghum has very efficient selective
mechanisms for the avoidance of a Na-enrichment in the
symplast (Koyro and Stelzer, 1988, Pei and Zhang, 1995).
The export of sodium, against a gradient of the
Na-concentration, from the cytoplasm into the nutrient
solution, into vacuoles, or into xylem element vessels
against a gradient of the Na-concentration is an energy
consuming process.
As shown for the halophyte Spartina townsendii, the
tolerance of high Na concentrations in leaf, shoot or root
minimizes the energy demand. In contrast to Spartina,
the salt-excluding Sorghum species needs metabolic energy
for the osmotic adaptation to organic substances, for a
highly selective K-uptake and probably for the recirculation of Na from the shoot to the root.
Zea mays showed a lower P level in mitochondria of
root cells after salt stress (Stelzer, 1984). In the saltsensitive sorghum, bean and cotton, salinity leads to a
decrease in cytoplasmic and vacuolar P concentrations of
root tip cells probably as a result of leakage due to
increased membrane permeability (Greenway et al., 1992;
Karaki et al., 1995; Koyro, 1989; Martinez and LauchJi,
1993). It is possible that P-concentration limits the
ATPase activity and the salt tolerance of sorghum.
Further salinity experiments are needed with varying P
supply to investigate its influence on the salt tolerance in
the root tip.
To summarize: the results show ultrastructural changes
caused by an extreme energy-consuming Na-avoiding
mechanism with extremely high energy consumption, as
well as a decrease in cytoplasmic concentrations of essential elements such as Mg, P, S, and Ca, thus causing the
ultrastructural degradations, indicated, either directly (via
energy supply in mitochondria, homeostasis, selectivity
of K over Na) or indirectly (via enzyme conformation,
cytoplasmic hydration).
The shown physiological and structural changes demonstrated have the potential to provide a highly valuable
means of detecting early stages of NaCl stress, and may
also provide opportunities for screening different varieties
for their adaptation to salinity.
Acknowledgements
We gratefully acknowledge the permission of Professor H Moor
(ETH, Zurich) to use the high-pressure apparatus in his
laboratory. We thank Drs W Kaeser and R Stelzer for critical
discussions and Dr G Collier for checking the manuscript. This
study has been supported by the 'Studienstiftung des Deutschen
Volkes' through a scholarship awarded to H-W K.
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