Journal of Experimental Botany, Vol. 51, No. 344, pp. 595–603, March 2000
Cell wall adaptations to multiple environmental stresses
in maize roots
Birgit Degenhardt and Hartmut Gimmler1
Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl Botanik I, Universität Würzburg, Germany
Received 19 July 1999; Accepted 10 October 1999
Abstract
A municipal solid-waste bottom slag was used to grow
maize plants under various abiotic stresses (high pH,
high salt and high heavy metal content) and to analyse
the structural and chemical adaptations of the cell
walls of various root tissues. When compared with
roots of control plants, more intensive wall thickenings
were detected in the inner tangential wall of the endodermis. In addition, phi thickenings in the rhizodermis
in the oldest part of the seminal root were induced
when plants were grown in the slag. The role of the
phi thickenings may not be a barrier for solutes as an
apoplastic dye could freely diffuse through them. The
chemical composition of cell walls from endodermis
and hypodermis was analysed. Slag-grown plants had
higher amounts of lignin in endodermal cell walls when
compared to control plants and a higher proportion of
H-type lignin in the cell walls of the hypodermis.
Finally, the amount of aliphatic suberin in both endoand hypodermal cell walls was not affected by growing
the plants on slag. The role of these changes in relation
to the increase in mechanical strengthening of the
root is discussed.
Key words: Endodermis, exodermis, heavy metals, lignin,
phi thickenings.
Introduction
Plants have developed a variety of strategies and mechanisms to react to any change of their environment.
Anatomical and physiological adaptations allow better
growth under unfavourable conditions. The root system
is particularly affected by these changes because the root
is that part of the plant which is in direct contact to the
contaminated soil substrate. A number of studies have
shown the alterations of root architecture and structure
induced by a variety of stressful conditions (Neumann
et al., 1994; Shannon et al., 1994; Tsegaye and Mullins,
1994; Plaut et al., 1997). For instance, it is known that
plants respond to salt stress by a reduction in root growth
and length. A decrease in the diameter of wheat roots
and of the transectional area of cortical cells was observed
with heavy metal treatment (Setia and Bala, 1994).
Besides changes in external root morphology there are
also alterations in the fine structure of the root. It has
been shown (Shannon et al., 1994) that salinity promotes
suberization of the hypodermis and endodermis and that
the Casparian strip is developed closer to the root tip
than in non saline roots. In cotton seedling roots, the
formation of an exodermis can be induced by salinity
(Reinhardt and Rost, 1995). In addition, some plant
species develop cell wall thickenings in different root
tissues. These thickenings are modifications of the midportion of the radial cell walls and are therefore called
phi thickenings. They can either form a uniseriate or a
multiseriate layer (Guttenberg, 1968; Peterson et al., 1981;
Eschrich, 1995). Phi thickenings are formed on the
walls of certain cell layers in the root cortex of several
species of gymnosperms ( Taxaceae, Podocarpaceae,
Cupressaceae) (Guttenberg, 1968; Haas et al., 1976) and
a few families of angiosperms (Rosaceae, Geraniaceae,
Berberidaceae, Sapindaceae) (Peterson et al., 1981;
Praktikakis et al., 1998). Thickenings can also be formed
in the hypodermis (e.g. geranium, Haas et al., 1976). All
species reported in the literature are dicotyledons.
However, the occurrence of phi thickenings in roots of
monocotyledons has not yet been documented.
Bulk density, porosity and mechanical impedance are
other factors influencing root growth and extension
(Bennie, 1996). Increased mechanical impedance slows
root extension and can alter cell size and cell number in
the cortex (Goss and Russell, 1980).
1 To whom correspondence should be addressed. Fax: +49 931 888 6158. E-mail: [email protected]
© Oxford University Press 2000
596 Degenhardt and Gimmler
In this study, two different soil substrates were compared for their effect on the growth of corn roots: garden
mould was used as a control substrate and MSW (municipal solid waste) bottom incinerator slag was used as a
contaminated substrate including different sources of
stress like salinity, high pH, heavy metals, and high
mechanical impedance. MSW incinerator slag is used in
Germany in accordance with German law as support and
construction material (LAGA, 1994). In general, it is
used for roads, banks or parking lots as a 30–50 cm thick
top layer. The environmental compatibility of this application is under discussion.
The aim of this study was to investigate how tissues in
corn roots were affected by the presence of various sources
of stress. Therefore, not only the development and structure of the root, but also particular components of the
cell wall were examined.
Materials and methods
Plant material
Seeds of corn (Zea mays L. cv. Garant FAO 240) were obtained
from Asgrow GmbH, Bruchsal, Germany. Seeds were imbibed
in aerated water overnight and planted in pots (90 mm diameter)
filled with soil. Plants were grown in a greenhouse (20 °C,
16/8 h, light/dark) for 20 d, and watered daily with tap water.
Soil substrates
Processed MSW slag, obtained from a MSW slag processing
plant Würzburg (CC Reststoff GmbH, Germany), was used as
a substrate expected to induce various kinds of stresses (alkaline
and salt stress, heavy metal stress) on plant roots.
In order to distinguish the alkaline stress from the salt stress
of the slag, the salt burden of the slag was removed by washing
with a 3-fold volume of water. For this purpose a defined
volume of slag was put into an Erlenmeyer flask, the same
volume of distilled water was added and the mixture was
shaken for 1 h. Then the mixture was allowed to settle, filtered
and the solid was resuspended with fresh water. This procedure
was repeated three times. The supernatant was discarded, the
slag was dried at 105 °C and stored until use.
A garden mould composed of organic soil, peat and sand in
a ratio of 452:1, by vol. was used as a reference soil. Before use
the organic soil was steam-heated (20 min, 90 °C ) to kill fungi
and weed seeds.
Element analysis
The elemental composition of the municipal incinerator slag
and the garden mould was determined after homogenization of
samples and pressurized extraction in ultra grade 65% HNO
3
(170 °C, 10 h) by ICP emission spectroscopy (ICP Jobin 70
plus, ISA GmbH, Grasbrunn, Germany).
Soil physical properties
Mechanical impedance was determined by a penetrometer ( Ele,
Great Britain). For determination of the bulk density (mass of
dried soil per unit volume) the soil substrates were dried at
105 °C, placed in a calibrated glass cylinder and weighed.
Soil solution
Soil solutions were obtained by negative pressure filtration with
suction cups (ceramics suction cups, Oekos, Göttingen,
Germany).
Microscopic techniques
For all microscopic investigations sections were made in the
upper third of the primary roots. By reason of different root
growth of slag and control plants, the whole root was divided
into three parts. For slag roots, the upper third is 1–5 cm from
the root base, for control roots 1–15 cm. Before use, root
segments were fixed for 24 h in a phosphate buffer (10 mM,
pH 7.4) containing 3.7% (w/w) formaldehyde. Sections of 20 mm
were cut at −25 °C using a cryomicrotome (Cryostat H 500 M;
Microm) and examined by an Axioplan microscope (Zeiss,
Oberkochen, Germany) equipped with an Osram HBO 50 W
mercury lamp and Zeiss filter sets (exciter filter 365 nm,
dichroitic mirror FT 395, barrier filter LP 395).
Tracer application
To test the permeability properties of the phi thickenings, a
berberine–thiocyanate tracer procedure was applied ( Enstone
and Peterson, 1992). The chemicals necessary for the experiment
were obtained from Sigma (Deisenhofen, Germany). The
movement of the fluorescent dye was observed on sections
under blue light using an axioplan microscope (Carl Zeiss,
Oberkochen, Germany).
The Casparian bands of endodermis and hypodermis were
visualized by treating root sections with 0.1% berberine
hemisulphate for 1 h, rinsing the tissue several times with water
and transferring to a 0.5% toluidine blue staining solution for
30 min. Toluidine blue is used instead of aniline blue. The tissue
was rinsed once again with distilled water, then placed on slides
in mounting medium (0.1% FeCl in 50% glycerol ) and viewed
3
under violet fluorescence excitation (Brundrett et al., 1988).
Isolation of hypodermal and endodermal cell walls
The isolation and purification procedures of cell walls of the
hypodermis and endodermis have been described in detail
previously (Schreiber et al., 1994). Roots were cut into segments
of approximately 5 cm length using a razor blade. Root
segments were incubated at room temperature in a citric buffer
solution (10−2 M, pH 3) containing cellulase (Onozuka R-10,
Serva, Heidelberg) and pectinase (Macerozyme R-10, Serva,
Heidelberg). Sodium azide was added to prevent microbial
growth. After 10–15 d of enzymatic digestion, hypodermal and
endodermal cell walls were separated by means of two forceps
and a binocular microscope (SZ 30, Olympus, Hamburg).
Determination of lignin and suberin
Thioacidolysis: To detect the biopolymer lignin, isolated cell
wall material was subjected to the thioacidolysis-procedure
(according to Lapierre et al., 1991). Cell wall isolates were
stirred in a mixture of BF etherate (Merck, Darmstadt,
3
Germany), ethanethiol (Fluka, Neu-Ulm) and dioxane in argon
atmosphere at 100 °C for 4 h. The reaction mixture was allowed
to cool, diluted with 2 ml water and extracted three times with
3 ml of CHCl containing 20 mg dotriacontane (Fluka) as
3
internal standard. The combined organic phases were dried
with Na SO .
2 4
Transesterification: The degradation of cell wall material for
detecting suberin was carried out according to the transesterification method ( Kolattukudy and Agrawal, 1974). 0.5–1 mg
Corn roots and environmental stress 597
of isolated cell wall material was added to 1 ml of a 10%
BF /methanol solution (Fluka). The reaction mixture was
3
heated to 70 °C for 24 h. After cooling, the solution was
removed from the cell wall sample and collected. The cell walls
were washed three times with 1 ml CHCl containing 20 mg
3
dotriacontane (Fluka) as internal standard. The chloroformic
and methanolic solutions were combined and washed twice with
2 ml saturated sodium chloride solution. The organic phase was
separated and dried with Na SO .
2 4
Gas chromatography and mass spectrometry
Gas chromatographic analysis and mass spectrometric identification of the reaction products obtained by thioacidolysis and
transesterification were performed as described in detail previously (Zeier and Schreiber, 1997). Separation and quantitative
sample analysis were achieved by a gas chromatograph (HP
5890 Series II gas chromatograph, Hewlett-Packard, California,
USA) equipped with a flame ionization detector. Qualitative
sample analysis was carried out on a gas chromatograph (HP
5890 Series II gas chromatograph, Hewlett-Packard) coupled
with a quadrupole mass selective detector (HP 5971 mass
selective detector, Hewlett-Packard). Before injecting, samples
were derivatized using BSTFA (N,N-bis-trimethylsilyltrifluoroacetamide, Machery-Nagel, Düren).
Analysis of amino acids
Cell wall isolates were hydrolysed with 1 ml 6 N HCl (130 °C,
24 h, argon atmosphere). After filtration the hydrochloric acid
was removed (20 mbar, 40 °C ), the residue washed twice with
1 ml distilled water and the aquatic solvent was evaporated
again. Then 200 ml sample buffer (0.1 M lithium citrate,
68.5 mM citric acid, 20 mM bis-(2-hydroxy-ethyl )-sulphide,
pH 2.2) was added. Amino acids were analysed by HPLC
(LC 5001 Biotronic, Eppendorf, Hamburg).
Results
MSW bottom slag compared to garden mould was characterized by its high Ca, Fe, Na, Cl, S, and B content
( Table 1), its high amount of heavy metals ( Table 2) and
its low N content ( Table 1). When compared to garden
mould the metals were 25–200-fold enriched (copper
Table 1. Major elements of MSW incinerator slag, washed slag
and garden mould
Si
Ca
Fe
Na
Mg
K
Cl
P
S
N
B
Content (g kg−1)
Garden mould
Washed slag
Slag
280–355
15–17
12–23
0.2
4.2–5.3
5.8–7.1
0.1
0.87–1.4
0.36–0.75
1.1–5.2
0.02
138–165
69.8–106
76.9–96.5
3.23–4.24
6.03–10.4
2.59–3.73
n.m.a
2.2–3.3
3.7–5.4
0.02–0.17
0.2
138–165
99–126
69–100
7.7–15
9.4–13
5.1–10
5
2.2–5.1
6.3–8.1
0.037–0.2
0.2
an.m., Not measured.
Element
Content (mg kg−1)
Cd
Cr
Cu
Ni
Pb
Zn
Garden mould
Slag
0.2
19–45
13–30
11–20
26–35
97–170
10
320–520
1260–3600
190–600
800–1600
3260–5900
(60–180-fold ), cadmium (50-fold ), lead (25–50-fold),
and zinc (25–50-fold )). A closer estimation of the phytotoxicity of this substrate is to assay the availability of
these elements in the soil solution. In Table 3 the chemical
composition of soil solutions obtained by suction cups is
shown. The solutions exhibited strong alkaline pH values
for slag and washed slag while the pH of the soil solution
of garden mould was only slightly alkaline. Even though
the amount of P and Fe was high in slag, these elements
were hardly detectable in the soil solution, due to the
insolubility of phosphate and iron compounds at alkaline
pH. Furthermore, the electric conductivity of the slag soil
solution indicated a 10 times higher concentration of
solutes than in the reference soil solution. The main
difference between slag and washed slag was the lower
content of soluble salts (alkali-chloride and -sulphate) in
washed slag. Details about the chemical composition of
the soil solution of slag and other properties of MSW
Table 3. Chemical composition of the soil solutions obtained by
suction cups
Characterization of the soil substrates
Element
Table 2. Heavy metal content of garden mould and slag
Content in substrate (mg l−1)
pH
Conductivity
(mS cm−1),
20 °C
Al (mg l−1)
B (mg l−1)
Ca (mg l−1)
Fe (mg l−1)
K (mg l−1)
Mg (mg l−1)
Mn (mg l−1)
Na (mg l−1)
Cd (mg l−1)
Cr (mg l−1)
Cu (mg l−1)
Ni (mg l−1)
Pb (mg l−1)
Zn (mg l−1)
Cl− (mM )
NH+ (mM )
4
NO− (mM )
3
PO3− (mM )
4
SO2− (mM )
4
Garden mould
Slag washed
Slag
7.4–7.9
0.931–2.48
8.1–8.4
3.3–6.7
8.4–9.0
21.4–33.3
0.02–0.06
0.22–0.47
81.2–170.7
0.043–0.122
98.9–322
17.3–34.3
0.0025–0.0197
24.2–77.4
n.d.–0.008
n.d.
0.038–0.111
n.d.
n.d.
0.073–0.80
2.64–6.08
16.5–34.2
4.8–10.98
0.3–1.6
5.4–6.5
n.d.a
1.88–2.71
636–829
n.d.–0.029
160–302
17.4–20.6
0.048
683–1150
n.d.–0.021
0.0146–0.0324
0.07–0.097
n.d.
n.d.
0.03–0.099
9.7–55.5
13.9–25.3
0.4–0.5
n.d.–0.04
19.5–23.2
0.30–0.95
2.8–3.9
881–1142
n.d.–0.03
1304–2117
74.8–96.9
0.013–0.037
3716–6337
n.d.–0.04
0.22–0.47
0.36–0.49
n.d.
n.d.
0.0025–0.043
82–204
21.5–130
n.d.
n.d.–0.4
26.4–37.6
an.d.: Not detectable.
598 Degenhardt and Gimmler
slag are listed (Fuchs et al., 1997). MSW slag is a rather
inhomogeneous substrate ( large differences in size of soil
particles, 40% of slag particles are larger than 7 mm in
comparison to 15% of the control soil ). For a description
of a soil substrate some physical properties should be
mentioned as well. Compared to garden mould
(0.07 MPa) the mechanical impedance of slag is increased
about a factor of three (0.2 MPa). Bulk soil density of
slag (1.14 g cm−3) was higher than that of garden mould
(0.92 g cm3).
Modifications in root architecture and structure
The effect of culture in slag was first a reduction of root
growth. Root length was reduced by 50% by cultivation
on slag compared to garden mould ( Table 4). However,
the root5shoot ratio was increased for slag plants by a
factor of 2. Root diameter was also affected. Roots grown
in MSW slag were 14% thicker. Microscopic examination
revealed that the increase of the root diameter was due
to an increased thickness of the cortex ( Table 4).
In roots of slag-grown plants intensive U-shaped thickenings of the inner tangential wall of the endodermis
(Fig. 1A) were observed. In the tertiary state of development endodermal cells deposit lignified cell wall material
onto the suberin lamella. These deposits were pronounced
more intensive in slag-cultivated roots (0.5±0.08 mm)
than in control roots (0.32±0.04 mm) (Fig. 1B). Another
phenomenon detected during the slag-cultivation of corn
were cell wall modifications like phi thickenings in the
radial cell walls of the rhizodermis ( Fig. 1C, D). These
phi thickenings were present only on slag culture. The
development of phi thickenings during these experimental
conditions was variable, both longitudinal and tangential.
Thickenings were not formed continuously from the tip
to the base, but occurred only in the upper third, the
oldest part of the root. In addition, if present, phi
thickenings did not occur continuously in the rhizodermal
periphery of the root. Rather intermissions in thickening
formation were observed. Between 6 and 50 adjacent
rhizodermal cells formed clusters equipped with phi thickenings. Attempts to induce phi thickenings by treating
roots with 100–200 mM NaCl in hydroponic culture or
with sand as carrier material or by growing them in glass
ballotini of varying size to simulate mechanical impedance
Table 4. Growth parameters of shoot and root of corn grown on
slag or garden mould; values represent means±SD, n=10
Seminal root length (cm)
Root fresh weight (g)
Shoot fresh weight (g)
Root5shoot ratio
Root diameter (mm)
Diameter of the central cylinder (mm)
Garden mould
Slag
63.2±5.8
2.60±0.67
6.33±0.63
0.41
1.16±0.11
0.55±0.02
35.2±4.2
1.6±0.54
1.8±0.53
0.92
1.35±0.16
0.55±0.03
failed (not shown). Also hydroponic and aeroponic cultures of Zea mays did not induce phi thickenings in the
rhizodermis (Freundl et al., 2000).
To get more information about the importance of cell
wall thickenings, the permeability properties were tested
by application the apoplastic tracer berberine hemisulphate (Enstone and Peterson, 1992). Berberine moves radially into the root and forms crystals with potassium
thiocyanate. As shown in Fig. 1E the phi thickenings
were penetrated, but the inward movement of the dye
was stopped at the exodermis. The cell walls with phi
thickenings were permeable for the apoplastic dye in
contrast to the exodermal Casparian band. Figure 1F
illustrates the occurrence of the Casparian band in the
exodermis and endodermis and reveals that maize is an
exodermal species ( Enstone and Peterson, 1992).
Chemical composition of cell wall isolates
Lignin: Quantitative chromatographic results revealed
that isolated cell walls of the endodermal layer of slagcultivated plants contained higher amounts of lignin than
cell walls of control roots ( Table 5). By contrast, roots
grown in prewashed slag did not contain higher concentrations of lignin. Furthermore, variations in the lignin
components were studied. The biopolymer lignin is composed of the cinnamic acid derivates p-coumaryl, coniferyl
and sinapyl alcohols, which differ in the extent of methoxylation. According to the aromatic nucleus in the polymer
the terms guaiacyl (G), syringyl (S) and p-hydroxyphenyl
(H ) are used. The monomeric composition of lignin
showed a difference both between cell walls of the endodermis and hypodermis and between the cell wall material
at different culture conditions. Although there was no
effect of the growth substrate on the total lignin content
of hypodermal cell walls (Table 5) there were variations
in the proportions of the monomeric units of lignin. The
proportion of the H-monomer in hypodermal cell walls
was much higher than in endodermal tissue ( Fig. 2A, B).
Furthermore, growth on slag induced a higher proportion
of H-units in hypodermal cell walls than in the control
ones. In addition, hydroponically grown maize showed
proportions of H-units even lower than garden mould
( Fig. 2A). In endodermal cell walls the proportion of
G-units were increased and the proportion of the S-units
were decreased by growth on slag. G-units were the
abundant monomer in the endodermis. In the hypodermis
the proportion of the three monomers is more
homogeneous.
Amino acids: Hypodermal cell walls in roots of slagtreated maize plants exhibited a nearly 3-fold higher
amount of p-hydroxyproline than the roots of the control
plants ( Table 6). The amount of the amino acids threonine, proline and histidine were also increased in roots of
Corn roots and environmental stress 599
Fig. 1. Fluorescent microscopic investigation of root cross-sections of primary roots of 20-d-old maize plants. (A–D, G) Autofluorescence (FT 395)
of unstained sections, made by the aid of a microtome. (A) Cross-section of the root of a slag plant showing massive tertiary wall thickenings of
the endodermis. (B) Cross-section of a similar aged root of a control plant. The U-shaped thickenings of the endodermis are significantly smaller
compared to the slag-treated root ones. (C ) Cross-section of the root of a slag plant showing wall modifications in the radial cell walls of the
rhizodermis (100×). (D) Cross-section as in (C ), 1000×. (E ) Freehand cross-section through the root of corn grown on slag, treated with berberine
hemisulphate and potassium thiocyanate (FT 460). Arrowheads indicate that the dye penetrated the wall thickenings, whereas the inward movement
was stopped at the Casparian band of the hypodermis (see F ). (F ) Cross-section of a control root treated with berberine hemisulphate,
counterstained with toluidine blue and viewed with violet illumination. The Casparian bands of the endodermis and hypodermis were obvious while
the fluorescence of the lamellar suberin and lignin is quenched. (G) Cross-section of a control root showing the lack of phi thickenings.
600 Degenhardt and Gimmler
Table 5. Amount of lignin of hypodermal (HCW) and endodermal
(ECW) cell walls obtained by thioacidolysis
Values in mg lignin mg−1 dry weight. Mean values and SD from three
independent experiments.
Lignin content (mg mg−1)
HCW
ECW
Garden mould
Slag washed
Slag
26.8±9.0
20.0±5.6
24.5±0.37
21.85±5
24.4±2.2
27.7±5.3
Fig. 3. Sum of aliphatic suberin monomers obtained after transesterification of hypodermal (HCW ) and endodermal ( ECW ) cell wall
isolates. Values are means of three replicates, bars indicate SD.
slag-cultivated maize plants. Finally, the sum of all
detected amino acids in hypodermal cell wall material of
slag-cultivated roots was higher than in control roots.
Hydrophobic compounds: There was a significant higher
amount of suberin in the cell wall of the hypodermis than
in that of the endodermis ( Fig. 3). However, there was
no significant increase in the total amount of hypodermal
suberin when the plants were grown on slag. It was also
Fig. 2. Monomeric units of hypodermal (A) and endodermal (B) cell
walls obtained after thioacidolysis; H ( p-hydroxyphenyl ), G (guaiacyl ),
S (syringyl ). Mean values of three independent experiments, bars
indicate SD. (Data of hydroponics were taken from Zeier, 1998).
Table 6. Amino acid composition of acid hydrolysates from
hypodermal cell walls in mg amino acid per mg dry weight; values
represent means±SD, n=3
Amino acid
Hydroxyproline
Proline
Histidine
Threonine
Sum (of all
detected
amino acids)
Content (mg mg−1 DW )
Garden mould
Slag
0.15±0.04
0.88±0.15
0.26±0.035
0.89±0.10
15.5±1.9
0.47±0.054
1.30±0.14
0.36±0.03
0.92±0.075
18.63±1.51
Fig. 4. Composition of aliphatic suberin components (sum=100%) of
hypodermal (A) and endodermal (B) cell walls obtained after
transesterification. Values are means of three replicates, bars indicate SD.
Corn roots and environmental stress 601
tested, whether growth of corn on slag would alter the
composition of the suberin fraction. Figure 4 demonstrates that this was not the case. The proportion of
v-hydroxyacids (C –C ), dicarboxylic acids (C –C ),
16 30
16 26
carboxcylic acids (C –C ), alcohols (C –C ), and
16 26
16 24
2-hydroxyacids (C –C ) were roughly similar in hypod16 26
ermis and endodermis and within a given cell wall type
between the various treatments.
Discussion
Soil substrates
When MSW slag is used as soil substrate, plants experience various stresses: salinity, nutrient deficiency, heavy
metal toxicity, alkaline pH, and compaction. High
amounts of soluble salts, mainly alkali chloride and
-sulphate salts are expected to cause considerable salt
stress to plants (up to −0.5 MPa) while the low N content
induces nitrogen deficiency. The low nitrogen content of
slag is due to its origin by burning of waste at 800–
1000 °C. The main difference between crude slag and
prewashed slag is the amount of salt. When slag is washed
with a 3-fold volume of water, most of the salt burden is
removed because of the high solubility of alkali salts,
mainly chlorides and sulphates.
The high pH of the soil solution of slag, which causes
alkaline stress, produces additional nutrition deficiency.
Although the elements P and Fe were present in higher
concentration in slag than in garden mould, at alkaline
pH they are scarcely available to the plant. Another stress
arises from the heavy metal content. Even though the
amount of heavy metals in slag is very high, the availability for plants is reduced due to the alkaline pH and the
chemical binding form of the metals (sequential extraction
procedure according to Tessier et al., 1979; not shown).
Thus, the contribution of heavy metals to the total stress
on slag-grown roots may be low.
Furthermore, plant growth was influenced by the compaction of the soil. Slag exhibits a stronger mechanical
impedance for the root system to penetrate into the soil
than the control substrate. It was reported that the
extension rate of the root was reduced when an external
pressure of about 100 kPa was applied (Goss and Russell,
1980). Other authors published values about 1.5 MPa as
moderate impedance. However, a lower impedance may
also impede root growth when soil aeration is low
( Vepraskas, 1994). Typical bulk soil density of heavily
compacted soils is about 1.5 g cm−3 (Iijima and Kono,
1992). Comparing with these data from literature the
mechanical impedance of MSW slag could be valued as
rather moderate and not severe.
Root structure
By cultivation of corn in slag, various architectural and
structural changes were observed. The decrease in root
length and the increase in root diameter are typical
morphological changes for mechanically impeded roots
(Bennie, 1996). Morphological and anatomical changes
of roots of young maize were also observed when the
seedlings had to penetrate soil of high mechanical impedance (Hartung et al., 1994). Salinity stress also causes a
thickening of the root. The increased root to shoot ratio
corresponds to the fact, that plants invest more energy in
root than in shoot growth in order to increase absorption
of ions when they suffer nutrient deficiency, here N and P.
Even though Zea mays is an intensively studied material, nothing else has been reported in the literature
about the presence of cell wall modifications like phi
thickenings in this plant to date. However, phi thickenings
have already been described for several other species (e.g.
pelargonium, apple, Pyrus) as ordinary structures in cell
walls of root tissue. They have been described for varies
root tissues, but never for the rhizodermis. At least, no
special conditions were reported to be necessary to induce
them. However, a phi layer in Ceratonia siliqua L. has
been described, which was observed when plants were
grown in soil but not in perlite (Praktikakis et al., 1998).
In the case of Zea mays, phi thickenings were only
detected in slag culture. Therefore the properties of the
soil substrate were an important parameter for the presence or absence of such cell wall thickenings. The variation in the frequency of phi thickenings may be attributed
to the inhomogeneity of the soil substrate. MSW slag is
a heterogeneous substrate. Before use, rough particles
(particle size >2 cm) were removed, but the bulk fraction
still contains fragments of glass or metals and the particle
size varies considerably. Thus, the bulk soil density could
differ locally. Therefore, roots could be influenced only
by stages in their extension. The exact reason for the
presence of phi thickenings in maize grown on slag is not
clear, but a relationship between the supporting role of
the wall thickenings and the mechanical impedance of the
soil substrate is very likely. Furthermore, the fact that
the thickenings do not function as a barrier for the
apoplastic movement of solutes and that they are not
present around the whole root circumference suggest that
they have no relevance as a barrier for solutes in the
rhizodermis. This is a further argument for the mechanical
supporting role, as suggested by the literature
Chemical composition of cell wall isolates
The chemical composition of hypodermal and endodermal cell walls were compared with respect to the
amount of lignin, amino acids of cell wall proteins and
hydrophobic compounds, because knowledge of the
chemical composition of cell wall material can be usefully
used to interpret the function of structural changes.
Referring to the hypothesis of mechanical adaptation of
roots grown in slag, the higher amount of lignin of the
602 Degenhardt and Gimmler
endodermis is a further argument. The incrustation of
the cell walls with lignin is reported to provide mechanical
stability for root architecture. The tight binding of lignin
to the cellulose-fibrils gives high static properties to the
cell walls (Richter, 1996).
Discussing the differences in lignin composition,
H-enriched lignin is proposed to be stress lignin (Monties
and Chalet, 1992) or lignin associated with compression
wood as in gymnosperms (Campbell and Sederoff, 1996).
A higher proportion of H-units produces a more condensed lignin because of more intermonomeric C–Clinkages. This functional adaptation of the hypodermis
could be related to the fact that the hypodermis is the
external sealing tissue of the root, which is in direct
contact with the surrounding rhizosphere and its microorganisms. This fact supports the hypothesis of the contribution of the hydroxyphenyl component to the hardening
of the cell walls.
Cell walls may contain both structural proteins and
enzymes. The amino acid composition of these peptides
can be analysed after acid hydrolysis. After the pretreatment of the cell wall material as carried out in this
investigation, proteins with a structural function are
assumed to remain in the cell wall because soluble or
poorly bound proteins would be removed. The amino
acid pattern of structural proteins varies from species to
species. Monocotyledons such as maize have a threonine–
hydroxyprolin-rich and a histidine–hydroxyproline-rich
glycoprotein ( Kieliszewski and Lamport, 1987;
Kieliszewski et al., 1990). Extensin is one member of a
class of hydroxyproline-rich glycoproteins (Richter,
1996). Extensin has frequently been associated with reinforcement of the cell wall network (Gladys et al., 1988).
The occurrence of extensin in the sclerenchyma could be
related to the mechanical function of this tissue in the
plant. In addition, extensin is absent from walls of young
root tissue (Gasparikova, 1992). It has been proposed
that extensin contributes, with other wall components, to
the tensile strength of mechanical cells.
The question arose whether or not cultivation of corn
on slag, with its high content of heavy metals, causes a
better exclusion of ion uptake through the apoplastic
pathway by higher amounts of suberin in the hypodermis,
particularly in the Casparian strips. The amount of suberin is a measure for the extent of the permeability of cell
walls for water and soluble ions ( Kolattukudy and
Agrawal, 1974). With regard to the chromatographic
results about hydrophobic compounds, it is obvious that
there is no significant increase in the total amount of
hypodermal suberin introduced by growing corn on slag.
This indicates that there is no induction of a tighter
control of apoplastic uptake by growing plants on slag.
On the other hand, the higher amount of suberin in the
cell wall of the hypodermis than in the cell wall of the
endodermis emphasizes the role of the hypodermis as a
physiological barrier for hydrophilic compounds.
Finally the results of investigations so far revealed that
alterations induced by cultivation in MSW slag were of
a structural nature. The roots responded to cultivation in
slag with mechanical strengthening. In future, efforts will
be exercised to find further physiological adaptations.
Some work dealing with peroxidase activity which is
involved in modifying cell wall components and detoxification of highly reactive molecules is in progress.
Acknowledgements
This investigation was supported by a grant from the
Graduiertenkolleg ‘Pflanze im Spannungsfeld zwischen
Nährstoffangebot, Klimastreb und Schadstoffbelastung’ (B
Degenhardt) and the SFB 251 ( TP A2, H Gimmler). We are
indebted to Lukas Schreiber for offering the opportunity for
fluorescence microscopic investigations and to Jürgen Zeier for
supporting gas chromatographic and mass spectrometric
analysis.
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