Suberin lamella development in maize seedling roots grown in aerated and stagnant conditions

12 Pages·2005·0.36 MB·English

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2005
284444455
Original Article
Suberin lamellae in maize roots
D. E. Enstone & C. A. Peterson
Plant, Cell and Environment (2005) 28, 444–455
Suberin lamella development in maize seedling roots grown
in aerated and stagnant conditions
DARYL E. ENSTONE & CAROL A. PETERSON
Department of Biology, University of Waterloo, Waterloo ON N2L 3G1, Canada
ABSTRACT Casparian bands, and often also suberin lamellae and ligni-
fied tertiary walls (see the recent reviews and references
Hypoxia can stimulate the development of a suberized exo-
therein by Enstone, Peterson & Ma 2003; Ma & Peterson
dermis in aquatic plants; however, its influence on this
2003). The endodermis normally matures in a very regular
aspect of terrestrial root development is sparsely docu-
pattern, and functions in water and nutrient uptake regula-
mented. To determine the effects of hypoxia on maize (Zea
tion, maintenance of root pressure, and protection against
mays cv. Seneca Horizon) roots, seedlings were grown in
pathogens and adverse environmental conditions, among
vermiculite (VERM), aerated hydroponics (AER), stag-
other things (Enstone et al. 2003). The exodermis is also
nant hydroponics with agar (STAG), or aerated hydropon-
capable of most of these functions (with the exception of
ics with agar (AERAG). The endo- and exodermis were
root pressure maintenance), but the course of its matura-
examined for wall modifications. Lateral root emergence
tion can be more irregular than that of the endodermis
and aerenchyma formation were documented qualitatively.
(Enstone & Peterson 1997; Hose et al. 2001).
The endodermal Casparian band formation was unaffected
In maize, exodermal development is ‘patchy’ (Von Gut-
by treatment. Endodermal and exodermal suberin lamella
tenberg 1968; Enstone & Peterson 1997). That is, deposition
formation was earliest and most extensive in VERM.
of lamellae in the exodermal layer is variable and appears
Suberization, especially in the exodermis of aerated treat-
to depend more on growth conditions than on the age of
ments, was depressed in all hydroponic media. In compar-
the cells. For roots grown in well-watered vermiculite, mat-
ison with AER, STAG exodermal lamellae were increased,
uration of exodermal cells occurs over a wide age range.
but endodermal lamellae were decreased. Since the suber-
Not all cells of a particular age will suberize at the same
ized exodermis forms a barrier to radial oxygen loss from
time, nor are the cells that do suberize evenly distributed
roots to the medium, its stimulation in STAG roots (which
around the layer (Enstone & Peterson 1997). Maize roots
also developed extensive aerenchyma) would help retain
grown in a well-aerated nutrient solution have vastly fewer
oxygen in the root. The reduction of endodermal lamellae
exodermal suberin lamellae than those of a comparable age
should facilitate oxygen diffusion into the stele. Clearly, the
grown in vermiculite (Enstone & Peterson 1998; Zimmer-
response to environmental conditions is variable within
mann et al. 2000). On the other hand, exposure of a portion
individual cortical cell layers. Additionally, the observed
of the root tissue to humid air accelerates exodermal suber-
patterns of lamellae, aerenchyma and lateral root
ization compared to constant hydroponic culture (Enstone
development indicate a tight radial co-ordination of root
& Peterson 1998; Zimmermann & Steudle 1998; Zimmer-
development.
mann et al. 2000). This suggests a strong environmental
influence on the maturation of this layer.
Key-words: Zea mays L.; aerenchyma; Casparian bands;
What effect does hypoxia, brought on by flooding for
endodermis; exodermis; hypoxia; radial oxygen loss
example, have on the maize exodermis? There have been a
(ROL).
number of studies on roots of wetland plants growing under
oxygen-deficient conditions that provide both indirect and
INTRODUCTION direct evidence that hypoxia stimulates exodermal devel-
opment (see review by Colmer 2003 and references
The exodermis is a common but not ubiquitous feature of
therein). Under oxygen-deficient conditions, aerenchyma,
plant roots adapted to a wide range of environments (Peru-
a highly porous tissue that forms in the central cortex as a
malla, Peterson & Enstone 1990). It forms as one or more
result of increasing levels of endogenous ethylene (ethene;
layers of the outer cortex, directly internal to the epidermis,
Drew 1997; Drew, He & Morgan 2000), facilitates oxygen
and shares a number of features with the endodermis, the
transport from the shoot to within a few centimetres of the
innermost cortical layer. In particular, both layers develop
root apex (Colmer 2003). An oxygen deficit in the rhizo-
sphere creates a sink for oxygen, leading to radial oxygen
Correspondence: D. E. Enstone. loss (ROL) from the root. Studies on ROL utilizing cylin-
E-mail: [email protected] drical Pt electrodes and, more recently, oxygen microelec-
444 © 2005 Blackwell Publishing Ltd
Suberin lamellae in maize roots 445
trodes have shown that in many species oxygen leakage is Experimental treatments
greatest in young, apical tissue and declines toward the base
Four treatments were initiated immediately upon transfer
of the root, indicating that these species develop a proximal
of the seedlings.
barrier to ROL (see, for example, Armstrong 1967, 1971;
Colmer et al. 1998; Visser et al. 2000; Armstrong et al. 2000; 1 Vermiculite treatment (VERM): continued growth in
McDonald, Galwey & Colmer 2001, 2002). Furthermore, vermiculite as described above.
applying additional hypoxic stress can induce or increase 2 Aerated solution treatment (AER): hydroponic growth
the effectiveness of the barrier. The position of this barrier in one-quarter-strength modified Knop’s solution with
coincides with that of the exodermis in Phragmites (Arm- continuous aeration with an air stone.
strong et al. 2000). Recently, it has been confirmed by 3 Stagnant solution treatment (STAG): hydroponic growth
correlative anatomical, biochemical, and oxygen in the same Knop’s solution with 0.1% (w/v) Difco Bac-
microelectrode work that the exodermis with suberin toAgar (Fisher Scientific Canada) incorporated into the
lamellae is the ROL barrier in several woody, Amazonian solution to prevent convective mixing of gases (Wieng-
floodplain species (De Simone et al. 2003). weera, Greenway & Thomson 1997). The nutrient-agar
As a part of an on-going study of the development of the mixture was initially flushed with air via an air stone, but
maize root exodermis, we examined root development and aeration was halted when the seedlings were installed. It
endodermal and exodermal suberin lamella deposition in was expected that the stagnant solution would become
aerated and stagnant nutrient solution culture compared progressively more hypoxic with time, similar to a natu-
with that in vermiculite culture. Aerenchyma formation is rally occurring waterlogging event. No attempt was made
compared between the various treatments and is related to to purge the solution with nitrogen to lower the oxygen
exodermal development and lateral root emergence. concentration rapidly or to seal the surface of the stag-
nant solution from the atmosphere as these do not real-
istically represent a natural event.
MATERIALS AND METHODS 4 Aerated agar solution treatment (AERAG): hydroponic
growth in the same Knop’s plus agar solution as STAG
Plant material and culture
but aeration was continued for the duration of the exper-
Maize kernels (Zea mays L. cv. Seneca Horizon, obtained iment. This treatment provided a control for the addition
from Ontario Seed Company, Waterloo, Canada) were ger- of agar to the solution.
minated in medium vermiculite watered with tap water (six
Each of these treatments was duplicated over time.
kernels per 150 mm plastic pot) in a growth chamber
(300 mmol m-2 s-1 PAR, 14 h day period, 23∞/18 ∞C, min
65% relative humidity). After 72 h, seedlings were gently Dissolved oxygen measurement
uprooted, root lengths were measured and the seedlings
with roots 30–40 mm long were transferred to one of two Oxygen levels and solution temperatures in the hydroponic
culture systems for application of experimental treatments. treatment systems were measured on the initial, third and
For further growth in vermiculite, seedlings were trans- sixth days of treatment with a MultiLine P4 Universal
ferred to large plastic pots (approximately 285 mm Pocket Meter (Wissenschaftlich-Technische, Weilheim,
diameter and 280 mm deep, 12 per pot) filled with Germany) equipped with a dissolved oxygen probe (Cel-
vermiculite that had been saturated with tap water and lOx 325). Duplicate measurements were taken approxi-
allowed to drain. The pots were then watered with one- mately 80 mm below the surface of the solution at the sides
quarter-strength modified Knop’s solution (Loomis & of the tanks.
Schull 1937) and returned to the growth chamber. These
plants were subsequently watered with tap water approxi-
Root growth and development assessment
mately every 2 d to ensure the medium remained moist.
For treatments applied in a hydroponic system, seedlings Three days after seeding transfer, three to four seedlings
were transferred to a previously described culture system per treatment were harvested to measure root length and
(Enstone & Peterson 1998). Briefly, a 32 L tank was subdi- determine growth rate. Six days after the treatments were
vided into two 16 L compartments, each with its own large initiated, four seedlings from each group were harvested
air stone. A thin sheet of styrofoam supported the kernels for assessment of suberin lamellae development. Final root
above the surface of the liquid while the roots extended lengths were measured, and then the roots were cross-sec-
down through holes in the sheet into the solution. A double tioned at 10 mm from the apex and from there every 20 mm
layer of cheesecloth over the top of the styrofoam and to the base of the root. These sections were stored in 75%
kernels kept the kernels moist. Root system shading was glycerine until they were briefly rinsed with water and
provided for the first 24 h with aluminium foil and thereaf- stained with Sudan red 7B (SR; Sigma, St. Louis MO, USA)
ter with black mesh shade cloth. Each hydroponically based to detect suberin lamellae (Brundrett, Kendrick & Peterson
treatment was carried out in a glasshouse under ambient 1991) in both the endodermis and exodermis. A scoring
lighting (e.g. 1525 mmol m-2 s-1 PAR at noon on a sunny day system to characterize SR staining in individual cells was
in April). similar to those used in previous studies (Enstone & Peter-
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
446 D. E. Enstone & C. A. Peterson
Figure 1. Daily maize root growth in each of
four growing conditions: VERM, vermiculite,
n = 7–12; AER, aerated hydroponics, n = 4–
15; STAG, stagnant hydroponics, n = 7–13;
AERAG, aerated hydroponics with agar,
n = 8–10 (see text for culture details).
son 1997, 1998): ‘0’ represented no SR staining (no detect- tertiary walls were also noted on the same samples to deter-
able suberin lamella); ‘1’ indicated some, but not complete mine if a relationship existed between the formation of
SR staining (a developing suberin lamella); and ‘2’ repre- these root features.
sented moderate to strong SR staining on all walls visible
in cross-section (a mature suberin lamella). All endodermal RESULTS
and exodermal cells were counted and scored according to
Characterization of the treatment solutions
this system. At each sample distance, usually three sections
were assessed this way. However, near the root apex, sec- The dissolved oxygen concentrations in the hydroponic-
tions were more difficult to cut cleanly enough to score the based treatments were similar at the time of transfer,
entire endodermis and exodermis. In some cases, only one namely day 0 (Table 1). In both aerated treatments, AER
or two sections per unit distance from the root tip were and AERAG, the dissolved oxygen levels remained at
counted. Rarely, none were counted. This problem primarily about the same levels. In the STAG treatment, the dis-
affected unstained (therefore unsuberized) root tissue in solved oxygen concentration dropped sharply between day
apical regions. Although counting and scoring of individual 0 and day 3 and remained at a low level for the balance of
cells was sometimes not possible, it was always possible to
determine that no staining reaction was present so that the
impact on data analysis for the zone of interest was minimal. Table 1. Dissolved oxygen concentration and temperature of
Initial experiments were conducted to determine daily treatment solutions were determined at the beginning, midpoint
root growth rate in each treatment. Seedings were set up and end of treatment period for each growth condition
under each experimental growth condition and daily
Time (days)
destructive harvests were conducted. The root lengths of
Growth condition
these seedlings were measured and the change in length (replicate) Measurement 0 3 6
determined on a daily basis by subtracting the initial root
length measured at transfer from the final length at harvest. AER (1)* [O] (mg L-1) 8.25 8.59 8.68
2
The resultant growth curves (Fig. 1) plus the experimental Temperature (∞C) n/a n/a n/a
AER (2)† [O] (mg L-1) 8.07 8.64 8.34
day 3 and day 6 root growth rate data were employed to 2
Temperature (∞C) 24.4 21.8 22.9
convert the ‘distance from apex’ measurements into ‘root
STAG (1)* [O] (mg L-1) 8.09 2.69 2.06
zone age’ measurements. This allowed a valid comparison 2
Temperature (∞C) n/a n/a n/a
of suberin lamella development between treatments. STAG (2) [O] (mg L-1) 7.20 3.08 3.96
2
Because of the root growth rates, data from several section- Temperature (∞C) 24.6 22.2 18.9
ing locations were frequently binned into the same tissue AERAG (1) [O2] (mg L-1) 7.74 9.18 8.31
age group. Statistical analysis was performed on the suberin Temperature (∞C) 26.5 21.5 22
AERAG (2)† [O] (mg L-1) 7.72 8.62 8.26
lamella data using the General Linear Means (GLM) pro- 2
Temperature (∞C) 26.7 21.7 22.8
cedure in SAS (SAS Institute Inc., Cary, NC, USA) at a
confidence level of a = 0.05.
Each value is the average of two experimental replicates (n = 8, in
Qualitative observations of the presence and relative
total). Numbers in parentheses after the growth condition name
positions of lateral roots, aerenchymatous lacunae, and indicate the replicate number, * or † indicates treatments that were
endo- and exodermal suberin lamellae, plus endodermal run simultaneously, and n/a indicates information is not available.
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
Suberin lamellae in maize roots 447
Figure 2. Illustration depicting the relative
growth of the roots in four growing conditions,
the development of endodermal and
exodermal Casparian bands and suberin
lamellae, and endodermal tertiary walls. The
youngest observed point of aerenchyma
lacunae and lateral root emergence is
illustrated. The scale to the left of the diagram
indicates root length in millimetres. The
individual scales to the right of each root
indicate the tissue age in days (d). Note that
this scale changes at day 3 for stagnant and
aerated nutrient agar. Sporadic formation of
suberin lamellae consisting of less than 10%
of the entire endodermis or exodermis is not
included in this figure. The earliest age of
lateral emergence is shown; however, the
range of age over which laterals can emerge
varied among treatment. See text for details.
Green lines, endodermal Casparian bands; red
lines, endodermal suberin lamellae; purple
lines, exodermal suberin lamellae and
Casparian bands; gold lines, endodermal
tertiary walls; blue bars, aerenchyma. Dashed
lines, incomplete development of structure in
layer; solid line, complete development of
structure in layer.
the experiment. Solution temperatures were similar 6 d (AER and VERM) or two linear growth rates from days
amoung treatments, with some variation reflecting the 0–3 and days 3–6 (STAG and AERAG). The resulting rela-
ambient levels in the glasshouse (Table 1). The unusually tionship between root length and tissue age, and the details
low day 6 temperature (18.9 ∞C) in STAG (2) was likely a of anatomical development in each treatment can be seen
result of a heating malfunction overnight in the glasshouse. in Fig. 2.
An additional AER treatment running in parallel with
STAG (2) also showed a temperature dip on day 6, with no
Development of suberin lamellae
significant difference between the 3 and 6 d growth rate
(data not shown), indicating that the duration of the low- Conversion of ‘distance from apex’ measurements to ‘age’
temperature exposure was not extended enough to impact allowed a direct comparison of the rates of suberin lamella
the experimental results. deposition between treatments containing roots of widely
Tests with a thermocouple psychrometer indicated no varying growth rates [25 mm d-1 (STAG) to 55 mm d-1
difference between the osmotic pressure of the AER, (AER)]. This comparison reveals striking differences in
STAG or AERAG treatment solutions at any sample point endodermal and exodermal development within and
during the experiment (data not shown). In addition, pH between treatment regimes (Figs 2 & 3). Statistical analysis
measurements taken from one AER and one STAG treat- showed a significant difference between ages within treat-
ment showed small increases in solution pH from day 0 to ment regimes, and for the interaction of age and treatment
day 6 (0.25 units in AER and 0.5 units in STAG). regime for mature suberin lamellae in both the endodermis
and exodermis. Observations on specific growth regimes
are detailed below.
Root growth rates
Suberin lamellae in both endodermis and exodermis
An initial experiment to determine root growth rate under developed earlier and more completely in roots grown in
the different experimental conditions yielded different vermiculite than in any of the other treatment media (Figs 2
growth curves over time (Fig. 1). Seedlings grown in ver- & 3; also see Figs 14a, 15a, 16 and 17a in Enstone & Peter-
miculite and AER exhibited linear growth from transfer to son 1997). In VERM roots, suberin lamella development
day 6. AERAG seedlings exhibited a slightly sigmoidal was detected in 1-day-old tissue. Lamella development pro-
growth curve with the change in the curve at day 3. STAG ceeded more quickly in the endodermis than in the exoder-
roots initially grew at a rate similar to those in the other mis (Fig. 3a). In tissue older than 4.5 d, no passage cells
treatments, but this rate slowed after day 3, resulting in an were detected in the endodermis (Fig. 3a). Roots grown in
overall rate that was approximately 60% less that that of hydroponic treatments had significantly fewer suberin
the other three treatments. These differences were taken lamellae in both the endodermis and exodermis compared
into account when calculating the ages of the tissue sections to VERM roots (see Figs 12 & 13 in Enstone & Peterson
examined by utilizing either a single linear growth rate over 1998). Endodermal lamellae deposition began in 3.5- to 4.0-
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
448 D. E. Enstone & C. A. Peterson
Figure 3. Suberin lamella deposition in
endodermal and exodermal layer expressed as
a percent of cells with nil, developing, and
mature lamellae for each treatment. Sampled
zones have been converted to tissue age in
days to enable a clear comparison of
development between treatments. Youngest
tissue is at the apex and 6-day-old tissue is
basal but formed after transfer to the
treatment. (a)–(d), endodermal layers; (e)–
(h), exodermal layers. (a) and (e), VERM; (b)
and (f), AER; (c) and (g), STAG; (d) and (h),
AERAG. Solid black indicates mature suberin
lamellae, grey indicates developing lamellae
and white indicates no lamellae. There are
statistically significant differences in lamella
formation between exodermis and
endodermis, and between growth regimes. See
text for details. Note that there is no data for
0.2 d tissue in the STAG treatment because
these roots were growing too slowly to capture
tissue this young at 10 mm from the apex.
day-old tissues of AER-, STAG- and AERAG-grown roots. band plasmolysis provided supporting evidence for this
Surprisingly, fewer endodermal cells developed lamellae assumption.
under STAG conditions compared to AER and AERAG Endodermal Casparian bands were visible in the SR-
(Fig. 3). Greater differences were observed in exodermal stained sections where there were no lamellae to obscure
suberin lamella deposition among the nutrient solution them. In older tissue regions, the bands filled the majority
treatments. In STAG roots, lamella formation began in of the length of the radial wall (dash type bands, see
younger tissue compared to AER and AERAG roots. In 6- Enstone & Peterson 1997) whereas in the younger tissue,
day-old tissue, STAG roots contained far more mature they appeared as dots localized closer to the inner end of
lamellae (62% of cells) than either AER (4%) or AERAG the radial wall. Band plasmolysis was frequently seen in
roots (31%). sections containing only Casparian bands, particularly dot-
type bands. In very young sections where no bands were
visible, plasmolysis figures were completely detached from
Development of Casparian bands and
the cell walls, confirming a lack of bands. In all treatment
tertiary walls
regimes, endodermal Casparian bands formed prior to the
Based on the results of a previous study (Enstone & Peter- tissue reaching 1 day-of-age. Bands were seen in 0.2-day-
son 1997), it may be assumed that exodermal Casparian old tissue in AER and AERAG regimes and in older tissue
band development occurred shortly prior to suberin in VERM. The STAG root growth rate was so slow that
lamella deposition, although it was not possible to see the sections were taken too far from the tip to detect any 0.2-
bands in SR-stained sections. The occasional occurrence of day-old zones (Fig. 3).
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
Suberin lamellae in maize roots 449
a b
Figure 4. Cross-sections of maize roots grown under STAG conditions for 12 d showing aerenchyma lacunae development in relation to
lateral roots. Sections are stained with SR. Figures are a composite of several photographs to maximize the areas in focus. (a) Younger root
region with small lacunae between emerged lateral roots. At this stage, a pronounced cortical bridge is retained around the lateral roots.
(b) Older root region with more mature lateral root. Larger lacunae have developed and one is now directly adjacent to the lateral root
base, reducing the cortical bridge width. L, lateral root; X, xylem vessel; *, aerenchyma lacuna; single arrowhead, endodermis; double
arrowhead, exodermis. Magnification bar = 100 mm.
No tertiary walls were observed in the exodermis any of lacked this tissue, but some did occur at the bases of the
the roots (Fig. 2). However, such walls did develop in the roots (in tissues formed prior to transplanting) and in 5.0-
endodermis and their development was influenced by the to 6.0-day-old regions. The lacunae that were present were
growth media (Fig. 2). Development was earliest in VERM intermittent, infrequent, and small. In a few roots, they
roots, and somewhat later in AER roots. The occurrence of extended into tissue as young as 4.5 d. Of the three nutrient
tertiary walls was much reduced (absent in 75% of roots solution treatments, AER roots had the least aerenchyma,
examined) in AERAG roots compared to VERM and with 34% of roots lacking the tissue. In the balance of the
AER. In STAG roots, virtually no tertiary walls formed. samples, it occurred primarily in 6-day-old regions. In rare
cases, it was present in tissue as young as 4.5 d. More aer-
enchyma formed in AERAG roots; it was consistently
Lateral root emergence
present in 6.0-day-old zones and extended in some cases to
Roots grown in all treatment regimes developed lateral 4.0-day-old regions. STAG roots contained extensive aer-
roots (Fig. 2) but the treatment had minor effects on their enchyma (Fig. 4). Large and frequent lacunae formed in
emergence. In VERM roots, laterals emerged in a rela- older zones, and the tissue extended even into the youngest
tively narrow band consisting of a 3- to 3.5-day-old root zones sampled. This was the only treatment regime in which
zone. More variability was seen in AER roots, with emer- lacunae developed prior to exodermal or endodermal
gence points ranging from 2.5 to 4 d. In agar-treated roots, suberin lamellae.
the point of lateral emergence ranged from 3 d (AERAG)
or 4 d (STAG) to 6-day-old tissue, suggesting that the agar
Correlative development of endodermis,
treatment may have slowed the emergence of some of the
exodermis, lateral roots and aerenchyma
primordia. It was not possible with the methods used in this
study to determine if the exact age of the primary root Regardless of the medium in which they were growing,
tissue in which primordia were initiated was altered by the roots typically displayed certain relationships among devel-
treatment regimes. However, in the sections examined, the opmental features. Many of these are illustrated for a typ-
youngest tissue observed with unemerged primordia was ical root grown in vermiculite (Fig. 5). The exodermis
1.5 d in VERM and AER, 1.0 d in AERAG and 0.5 d in matured in a patchy manner, with some unsuberized cells,
(STAG) suggesting that initiation was not delayed. It some developing suberin lamellae, and some mature
would be premature to conclude that agar-bearing solu- suberin lamellae in the layer over a 4-day interval (Fig. 5b–
tions stimulate an earlier primordium initiation, as techni- f). Exodermal cells overlying unemerged lateral roots vir-
cal difficulties associated with sectioning the 0.2 m-0.5- tually always lacked suberin lamellae, and endodermal pas-
day-old tissue reduced the number of sections observed in sage cells were frequently radially aligned with the
some treatments. unsuberized cells in the exodermis. This was not an inflex-
ible rule for all passage cells but, in most sections observed,
at least some of the passage cells opposed unsuberized
Aerenchyma formation
exodermal cells. Exodermal cells surrounding emerged lat-
Aerenchyma development was strongly influenced by erals remained unsuberized longer than other cells in the
growth medium (Fig. 2). VERM-grown roots typically layer. It was extremely rare to see suberized exodermal
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
450 D. E. Enstone & C. A. Peterson
Figure 5. Diagrammatic representations of sections taken from
regions along a single, vermiculite-grown maize root, showing
positional relationships of endodermal and exodermal
suberization, lateral roots and aerenchyma, and the variability that
occurs in these relationships. Keep in mind the three-dimensional
aspect of the roots when looking at these two dimensional images.
Patterns of suberization in the pictured tissue are partly influenced
by structures in adjacent sections. White areas, primarily cells with
no suberin lamellae; grey areas, cells with developing suberin
lamellae, often including a few cells lacking lamellae and a few with
mature lamellae; black areas, primarily cells with mature suberin
lamellae. (a) In this 6-day-old section, all cells of the endodermis
and exodermis have a complete suberin lamella; they have formed
immediately adjacent to the emerged lateral. A few aerenchyma
lacunae are clustered on opposite sides of the cortex. Neither
cluster is near the lateral. This is the only zone with aerenchyma
in this root. (b) Passage cells are present in the 4-day-old
endodermis, including areas adjacent to the emerged lateral. A
large cluster of passage cells is opposite an exodermal disruption
caused by another lateral that has emerged obliquely. Unsuberized
exodermal cells are positioned adjacent to the emergence points
of both laterals. A zone of intermingled unmodifed and mature
cells is opposite some of the endodermal passage cells. (c) In this
3.5-day-old section, there are only a few endodermal passage cells,
none adjacent to the emerged lateral. Two of these passage cells
are opposite unsuberized exodermal cells; the rest face completely
suberized cells. The lateral is flanked by unsuberized exodermal
cells. (d) This 3-day-old section is a classic demonstration of the
patchy nature of exodermal suberization. Note that the
endodermal passage cells face the unsuberized exodermal cells. (e)
The just-emerging laterals in 2.5-day-old tissue are flanked by
endodermal and exodermal cells lacking lamellae. Several of the
endodermal passage cells are not radially aligned with unsuberized
exodermal cells. (f) The uneven distribution of exodermal cells
with suberin lamellae is clear even in 2.0-day-old tissue. (g) One-
day-old tissue has not begun to develop suberin lamellae in either
endodermis or exodermis.
Lacunae in the aerenchyma typically occurred internal to
suberized exodermal cells and rarely occurred under
unsuberized exodermal cells. However, in stagnant condi-
tions aerenchyma developed prior to exodermal suberin
lamellae. In vermiculite-grown roots, the exodermis became
almost entirely suberized in the older zones examined
(Figs 3e & 5a); however, aerenchyma was seen in only some
of these roots and then only in 5.0- to 6.0-day-old tissue. In
younger zones, aerenchyma lacunae did not occur near
emerging and emerged lateral roots (Fig. 4a). This ‘zone of
inhibition’ occupied one-sixth to one-quarter of the total
cortex including the lateral root. Lacunae could develop
directly adjacent to laterals in older tissues (Fig. 4b).
Spatial aspects of root development
cells immediately adjacent to a lateral root if large portions The results above have been described on a temporal basis.
of the exodermis were still unsuberized. In the vermiculite However, due to the difference in growth rate between
treatment, in which the exodermis became more com- roots in stagnant agar and other media, the spatial view of
pletely suberized than in other treatments, by day 6.0 suber- the root is different (Fig. 1). Externally, the main difference
ized exodermal cells were present adjacent to the laterals would be the shorter length of the roots grown in stagnant
(Fig. 5a). However, this situation was rare or non-existent solution and emergence of laterals closer to the tip. These
in tissues that were 4.0-day-old or younger (Fig. 5b–g). and the internal differences are summarized in Fig. 2.
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
Suberin lamellae in maize roots 451
DISCUSSION VERM-grown roots followed a sequence of early forma-
tion of bands and lamellae in both endodermis and exoder-
Media parameters and root growth rates
mis, leading to the complete sealing of both layers with
The treatment media in this study were selected to compare suberin lamellae (no endodermal passage cells or non-
root growth under varying environmental conditions. One suberized exodermal cells) in the oldest root zones. This
solid, but highly porous medium, VERM, was compared contrasted strongly with the extremely reduced presence of
with three solution-based media, AER, STAG and endodermal suberin lamellae and the lack of any exoder-
AERAG. The highly porous VERM and the pump-aerated mal modification in the AER treatment. These patterns of
AER and AERAG delivered a constant level of oxygen to development have been observed previously (Enstone &
the roots whereas the non-aerated STAG provided a rapid Peterson 1998; Zimmermann et al. 2000) and have been
drop in oxygen concentration (on average from 7.65 to exploited to allow biochemical characterizations of Caspar-
3.01 mg L-1 within 3 d). Oxygen concentrations at the latter ian bands and suberin lamellae (Zimmermann et al. 2000).
level are low enough to presume hypoxic stress is acting on Very little aerenchyma developed in the AER treatment
the root, although predictions cannot be made about the suggesting that, with the lack of exodermal modification,
root internal oxygen status based on the bulk solution oxy- oxygen acquisition from the medium (and perhaps also
gen concentration (see Verslues, Ober & Sharp 1998 for ethylene flux away from the root) was adequate to avoid
measurements in maize; also see reviews by Armstrong triggering a stress response.
et al. 1991; and Armstrong & Drew 2002 for detailed dis- The developmental changes in STAG-grown roots were
cussion of the difficulties of predicting root internal aeration as striking as the reduction in root growth rate. Hypoxia
related to external oxygen solution concentration, bound- induced exodermal Casparian band and suberin lamellae
ary layer thicknesses, root diameter, tissue porosity, and development in the older regions of the root. The amount
respiratory demands). No difference was noted in osmotic and extent of this development was not as great as in
pressure between solutions with agar (AERAG, STAG) and VERM roots; however, it was significantly greater than in
that without (AER), and pH changes in the AER versus either of the aerated solution treatments. A surprising
STAG solutions over the growth period were small. Thus, result was the reduction of lamella formation in the endo-
the main influences on root growth were media composition dermis of STAG roots compared to the aerated treatments.
(solid versus liquid) and oxygen concentration. These results clearly show that suberin lamellae formation
Root growth rates were similar in the three aerated treat- in maize endodermis is keyed more to environmental influ-
ments, with solid versus liquid media not causing any major ences than is Casparian band development. Further, the
effect on root elongation. As might be expected, the impo- same stimulus may, under some circumstances, provoke an
sition of hypoxia strongly reduced the growth rate in opposing response in the endodermis compared to the exo-
STAG-grown roots, resulting in roots that were about one- dermis. Substantial aerenchyma development occurred in
half the length of those grown in the other regimes. In STAG, far more than in the other treatments, an expected
addition, it could be seen that root growth rate was related result because hypoxia is known to induce this tissue via
to the level of oxygen; the roots grew faster during the first ethylene (see Drew 1997; Colmer 2003).
half of the treatment period while the oxygen concentration The AERAG treatment was implemented to examine
was dropping in comparison with the latter half when the the effect of supplying agar to the nutrient solution. There
oxygen concentration was consistently low. Ethylene prob- was no measurable difference between the osmotic pres-
ably plays a role in this growth reduction along with the sures of the solutions with and without agar, probably
oxygen deficit; hypoxia induces ethylene, which in turn because the pressures were at the detection limit of the
reduces root elongation (Drew 1997; also see Fig. 1 in instrumentation. Thus, any water stress due to the osmotic
Gunawardena et al. 2001). effect of agar added to the AERAG solution is negligible.
AERAG-grown roots produced small amounts of exoder-
mal suberin lamellae in 6-day-old tissue. This stimulation of
Anatomical differences resulting from
development compared to AER roots is probably due to
environmental alterations
an increased width of the boundary layer at the root surface
The altered environmental conditions produced an extreme which would increase the diffusion path of oxygen to the
change in root growth rate only under one treatment root and result in a very mild hypoxic stress. The diffusion
regime (STAG); however, roots from all treatments showed path of ethylene from the root would also be increased.
some striking anatomical differences. For example, endo- Support for this hypothesis is provided by the small
dermal suberin lamellae and tertiary walls, and exodermal increase in aerenchyma produced in AERAG roots.
Casparian bands and suberin lamellae varied with environ-
mental conditions. In contrast, endodermal Casparian
Environmental control of exodermal and
bands were not affected. In all treatments, these bands
endodermal maturation
formed near the root apex, usually within the first half-day,
suggesting that the programme regulating their formation The first question prompted by the results is, what stimuli
follows a strict developmental sequence that is not sensitive are triggering the various responses among the treatment
to the environment. groups? Two potential stimuli are hypoxia and drying
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
452 D. E. Enstone & C. A. Peterson
stress. A third potential stimulus, the accumulation of phy- strategy is successful at the oxygen concentrations provided
totoxins (primarily organic acids produced by decomposing in the AER treatment. Hypoxia, however, appears to be
plant material in hypoxic environments which have been the signal for exodermal suberin lamellae to form in STAG
shown to provoke a reduction in ROL (Armstrong & Arm- roots. The hypothesis that exodermal suberin lamellae and
strong 2001) have not been considered here because of the Casparian bands provide a barrier to radial oxygen loss
short experimental duration and small pH change over that (ROL) from the root into the rhizosphere (Armstrong
time. 1967; Colmer et al. 1998; Armstrong et al. 2000; Visser et al.
VERM is a well-aerated medium, providing an optimal 2000; Colmer 2003) has recently been confirmed in several
level of pO within the young maize root for growth and species (De Simone et al. 2003). A substantial body of work
2
metabolism (Verslues et al. 1998). [It should be noted that documents the existence of a peripheral barrier to ROL in
the vermiculite-grown maize roots examined by Verslues wetland plants that constitutively form an exodermis (e.g.
et al. (1998) were 80–100 mm long and, based on our pre- Phragmites, Armstrong et al. 2000; various grasses,
vious studies on maize root development, would be approx- McDonald et al. 2001, 2002) and the induction of such a
imately 4 to 5 days old (Enstone & Peterson 1997). Roots barrier in other species upon exposure to hypoxia (e.g. rice,
of this age have patchy exodermal suberin lamella devel- Colmer et al. 1998; various wetland species, Visser et al.
opment starting in the basal 10–20 mm (Enstone & Peter- 2000; Amazonian floodplain species, De Simone et al.
son 1997) and the balance of the root has no suberin 2003). Darwent et al. (2003) compared radial and longitu-
modification that could act as a barrier to ROL or to oxy- dinal oxygen profiles of non-aerenchymatous (grown under
gen uptake.] The high porosity of vermiculite may lead to conditions comparable to AER) and aerenchymatous
large airspaces directly impinging on the root epidermis, (grown under stagnant conditions) maize roots. Both types
which may then lead to small areas of evaporative water of roots showed a steep gradient in pO across the epider-
2
loss from the epidermis and a signal suggesting localized mal-hypodermal layers in the basal half of the root and
drying stress. Additionally, the location of water and nutri- aerenchymatous roots retained this steep gradient for an
ent uptake in VERM is limited to the regions of contact extensive distance further acropetally. This suggests stimu-
with the medium, leading to an increased overall resistance lation of a ROL barrier in stagnant-grown maize roots, in
to water flow into the root (see Discussion in Verslues et al. broad agreement with the suberization results of the cur-
1998); this may also lead to a drying stress compared to rent study. The pO gradient across the cortex–stele bound-
2
solution-grown roots. The roots responded by developing ary in non-aerenchymatous roots showed a reduction in
suberin lamellae in both layers to reduce water loss to the oxygen diffusion into the stele correlated roughly with
environment. Previous work has shown that root zones endodermal wall thickening. Data was not presented for
exposed to humid air will be stimulated to deposit exoder- the gradient into the stele in aerenchymatous roots, so it is
mal suberin lamellae and Casparian bands, plus endoder- not possible to look for correlations with the current suber-
mal suberin lamellae, compared to zones that remain in ization results (Darwent et al. 2003).
nutrient solution (Enstone & Peterson 1998). One advantage of a ROL barrier in combination with
In AER, AERAG, and STAG, drying stress is not an constitutive or induced aerenchyma is the conservation of
issue. However, hypoxia must be considered even under oxygen imported from the shoot in the oldest regions of the
aerated hydroponic conditions because of the relatively low root. Some of this oxygen can move to the root tip and
solubility of oxygen in water and because of the reduced support continued growth, to a limited extent, in an oxy-
delivery of oxygen to the rhizosphere in aqueous versus gen-deficient environment (Colmer 2003). In addition, a
gaseous media (e.g. Verslues et al. 1998; Colmer 2003). ROL barrier likely offers protection from ingress of phyto-
Hydroponically grown maize roots aerated vigorously with toxins which are formed in an oxygen-deficient, reducing
ambient air had reduced cortical oxygen concentrations environment (see review by Armstrong 1978). It appears
compared with vermiculite-grown maize (Verslues et al. that maize has the capacity to respond to hypoxia (such as
1998), and aeration with double the ambient level of oxy- would be imposed by flooding in the field) by forming exo-
gen was required to equal the levels of the vermiculite- dermal suberin lamellae (this study) and to develop a ROL
grown roots (Verslues et al. 1998). Thus, the AER- and barrier (Darwent et al. 2003). Direct correlative work with
AERAG-treated seedlings may have experienced a mild oxygen microelectrodes is needed to confirm this function;
hypoxic stress, whereas the level experienced by the STAG- however, it is logical to propose that a ROL barrier is
treated seedlings was far more severe. another putative function of the maize exodermis.
Secondly, what is the physiological significance of the If the increase in exodermal suberin lamellae in response
endodermal and exodermal response to the varying growth to hypoxia functions to conserve oxygen within the root,
conditions? It is possible that exodermal suberin lamellae then we can speculate that the decrease in endodermal
formation in AER roots is suppressed to increase oxygen suberin lamellae may assist the diffusional delivery of oxy-
diffusion into and ethylene diffusion out of the cortex. Sup- gen to the stelar tissue from the aerenchyma. To our knowl-
port for this idea comes from Malik et al. (2003), who have edge, the reduction in endodermal lamellar deposition has
shown that oxygen diffusion into the older regions of wheat not been described before, and it would be very interesting
roots grown with partial oxygen deficiency can help aerate to test whether or not stelar oxygenation is improved by
the root apices. The lack of aerenchyma suggests that the this mechanism.
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455
Suberin lamellae in maize roots 453
Co-ordination of cellular and tissue development barrier that help to oxidize the rhizosphere locally where
within roots the new root will be growing. A recent, extremely interest-
ing finding is the co-ordination of xylem poles, endodermal
Co-ordinated radial development between cells and tissues passage cells and unsuberized exodermis in Salix (Lux et al.
has been demonstrated previously for the endodermis and 2004). Neither lateral roots nor aerenchyma was present in
xylem; in many species, maize included, endodermal pas- the samples examined; however, in the majority of species
sage cells form in radial alignment with protoxylem ele- lateral roots are initiated in the pericycle adjacent to the
ments (Esau 1965). Maize root exodermis matures in a xylem poles. In a related vein, Konings & Verschuren
‘patchy’ (asynchronous) fashion (Von Guttenberg 1968; (1980) observed that cortical bridges were radially aligned
Enstone & Peterson 1997), but the pattern of cells with and with xylem poles in about 70% of observed instances in
without suberin forms no recognizable pattern when maize. There is some debate regarding the exact origin of
viewed in isolation. However, the current results show maize lateral roots. Bell & McCully (1970) observed the
clearly that there is a high level of co-ordination between initial divisions of the pericycle opposite the phloem in
lateral roots and cortex of developing maize roots. In some cultivar Seneca Chief, whereas Casero, Casimiro & Lloret
fashion, the development and emergence of lateral roots, 1995) detected them close to the phloem in an unnamed
the suberization processes of the exodermis and the endo- cultivar. Functionally, in maize this may not be important
dermis, and the regulation of programmed cell death lead- since the protoxylem and protophloem poles alternate in
ing to aerenchyma formation (Drew et al. 2000; close proximity around the circumference of the stele, and
Gunawardena et al. 2001) are all co-ordinated to leave emergent lateral root primordia typically span one phloem
intact spokes or bridges of parenchyma spanning the cortex and two xylem poles.
from the primordium to a cluster of exodermal cells that The patterns of co-ordinated development described
remain unsuberized. References to various combinations of above were seen in all treatments examined in the current
this development in various species, particularly lateral root work as well as in some preliminary work on aeroponically
primordia in conjunction with retained cortical bridges, are grown maize (unpublished results) indicating that the pat-
found in the literature (see Table 2), although none refer to terns are under strict genetic control whereas the levels of
all four features simultaneously. Co-ordination of the lat- expression of the components are modulated by environ-
eral roots, cortical bridges and unmodified exodermis has mental conditions. Little is currently known about the
been observed in Phragmites (Justin & Armstrong 1987; molecular regulation of development patterns in roots, espe-
Armstrong et al. 2000; Soukup, Votrubová & Cˇízˇková cially concerning the endodermis and exodermis. For an
2002), and it is believed that the unsuberized exodermal exception, see the study by Benfey et al. (1993) on the signal
‘windows’ provide a low resistance point of egress for the to initiate formation of the endodermal layer. It will be
emerging lateral (Armstrong et al. 2000; Soukup et al. 2002; especially fascinating to work out the signalling mechanisms
see also Armstrong 1971 for rice) as well as gaps in a ROL that underlie these co-ordinated patterns of development.
Table 2. Developmental correlations between root features
Xylem Lateral Endodermal Cortical Unmodified
Species poles roots passage cells bridges exodermal cells Ref.
Zea mays ¥ ¥ 4
¥ ¥ 3
Oryza sativa ¥ ¥ 2, 3
Phragmites sp. ¥ ¥ ¥ 1, 3, 7
Salix sp. ¥ ¥ ¥ ¥ ¥ 5
Carex nigra ¥ ¥ 3
Juncus effusus, ¥ ¥ 6
Glyceria maxima,
Nardus stricta,
Carex curta,
Narthecium ossifragum
The term ‘cortical bridges’ refers to intact cortical tissue along a radius from the endodermis to the exodermis flanked by aerenchyma
lacunae. Note that in all cases where aerenchyma is present in the roots, it is the lysigenous type. ¥ indicates the features which were observed
to be aligned in the species examined. 1, Armstrong et al. 2000; 2, Clark & Harris (1981); 3 Justin & Armstrong (1987); 4, Konings &
Verschuren (1980); 5, Lux et al. 2004; 6, Smirnoff & Crawford (1983); 7, Soukup et al. (2002).
© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 444–455