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ORIGINAL PAPER
Root anatomy of Pinus taeda L.: seasonal and environmental
effects on development in seedlings
Prem Kumar ÆStephen W. Hallgren Æ
Daryl E. Enstone ÆCarol A. Peterson
Received: 12 February 2007 / Revised: 1 June 2007 / Accepted: 9 August 2007 / Published online: 15 September 2007
Springer-Verlag 2007
Abstract The environmental and seasonal effects on
anatomical traits of Pinus taeda L. seedling roots were
studied in the laboratory in three contrasting root growth
media and also in typical outdoor nursery culture. Growth
media with lower water regimen and high penetration
resistance caused a reduction in lengths of the white and
condensed tannin (CT) zones and acceleration of devel-
opment of suberin lamellae in the endodermis. As a
possible counter to this reduction in zone lengths, second-
order laterals were produced closer to the tips of first-order
laterals. This suggested there may be an advantage to
producing more shorter roots under stressful conditions.
Under outdoor nursery conditions (June to mid-December)
the white zone was always a rather small part of the root
system surface area (4.5% in December), but it dominated
as a provider of cortical plasmalemma surface area (CPSA)
in contact with modified soil solution (65% in December)
because of its live cortex and capacity to increase nearly
three fold the amount of CPSA per unit root length. The CT
zone always provided most of the total root surface area
(80% in December). Although it had no live cortex, a few
cells of the CT zone endodermis remained non-suberized
passage cells, perhaps giving this major part of the root
system some capacity for ion and water absorption. A late
summer increase in CPSA was due largely to the rapid
production of mycorrhizae. Root systems were capable of
very rapid replacement of roots lost due to undercutting
and lateral root pruning. The great variation in CPSA per
unit root length contained in the white, mycorrhizal and CT
zones suggested a capacity to adapt rapidly to changing
conditions.
Keywords Condensed tannin zone
Cortical plasmalemma surface area Mycorrhizal roots
Passage cells Suberin lamellae
Introduction
Previous research based on careful anatomical work with
Pinus banksiana and Eucalyptus pilularis described the
root system as being composed of three root zones (white,
condensed tannin and cork) (McKenzie and Peterson
1995a,b). These zones were later observed in P. taeda
(Peterson et al. 1999; Enstone et al. 2001). The white zone
had: (1) living cortex, (2) endodermis with abundant non-
suberized passage cells, and (3) central stele containing
conducting xylem except at its extreme tip. The condensed
tannin (CT) zone was characterized by dead and dying
cortex and a decreasing number of endodermal passage
cells. The cork zone had significant secondary xylem and
phloem growth to facilitate axial transport, and a complete
ring of mature cork cells. The above-described system of
dividing roots into three zones has distinct advantages over
earlier studies where roots were considered to have two
zones, i.e., non-suberized and suberized (Addoms 1946;
Communicated by R. Hampp.
P. Kumar
Department of Botany, Miami University,
Oxford, OH 45056, USA
S. W. Hallgren (&)
Department of Natural Resource Ecology and Management,
Oklahoma State University, 022 Ag Hall, Stillwater,
OK 74078-6013, USA
e-mail: steve.hallgren@okstate.edu
D. E. Enstone C. A. Peterson
Department of Biology, University of Waterloo,
N2L 3G1 Waterloo, ON, Canada
123
Trees (2007) 21:693–706
DOI 10.1007/s00468-007-0162-y
Kramer and Bullock 1966), white and brown (Sands et al.
1982), non-woody and woody (Van Rees and Comerford
1990; Escamilla and Comerford 2000), or primary and
secondary (McCrady and Comerford 1998). The large
cortical plasmalemma surface area (CPSA) of the white
root zone provides a great potential for ion uptake, as it is
in direct contact with the modified soil solution (MSS) in
the apoplast of the cortex. Factors expected to influence
water uptake include death of the cortical cells, suberin
lamella development in the endodermis, and formation of
cork.
Our understanding of how woody roots develop can be
advanced by determining how the three zones are affected
by specific stresses, and by field soil conditions. To date,
the white, CT, and cork zone anatomies have been char-
acterized in roots grown in pouches, growth chamber pot
culture and in sandy soil in the field (Peterson et al. 1999;
Taylor and Peterson 2000; Enstone et al. 2001). The
objective of the present study was to determine environ-
mental and seasonal effects on the development of the root
zones of P. taeda seedlings. For the former, we studied
first-order lateral roots of 2-year-old P. taeda seedling roots
grown in three widely different conditions: mist chamber,
peat-vermiculite and loamy sand. For the latter, we studied
the entire root system, including higher order lateral roots
and mycorrhizae, of P. taeda first-year seedlings grown in
a nursery under operational cultural treatments over
6 months.
Materials and methods
Source of plant material
Seedlings of P. taeda L. were grown in the Oklahoma
State Forest Regeneration Center, Washington, Oklahoma
(3509013.5900 N latitude and 9727041.8800 W lon-
gitude) from seeds of genetically improved open-
pollinated Oklahoma sources according to operational
procedures.
Microscopy and histochemistry
Observations were conducted on fresh freehand sections.
Endodermal Casparian bands were visualized using the
berberine–aniline blue technique of Brundrett et al. (1988),
which causes the bands to fluoresce bright yellow under
ultraviolet (UV) excitation. Suberin lamellae in the endo-
dermis and cork cells were stained with fluorol yellow 088
(FY), mounted in 75% glycerol, and excited with UV light
(Brundrett et al. 1991), causing the lamellae to fluoresce
bright yellow.
The maturity of xylem tracheids in the tips of white
roots was tested using Cellufluor (Calcofluor White M2R),
a fluorescent dye that binds to cellulose in a 0.01% w/v
solution (Hughes and McCully 1975), using the method of
Peterson and Steudle (1993). When this solution was
applied to the severed basal end of a root tip only mature
tracheids conducted the stain and became bright blue under
UV illumination.
To measure the lengths of cortical and endodermal
passage cells, a novel clearing and staining technique was
developed. Roots were soaked in 10% Clorox
TM
bleach
(5.25% sodium hypochlorite) for 1–2 h until completely
clear of any traces of condensed tannin. Bleach was
removed by rinsing the cleared root segments 3–4 times
with tap water. Suberin lamellae in the endodermis were
stained with FY (Brundrett et al. 1991), and endodermal
passage cells were differentiated from neighboring suber-
ized endodermal cells by the lack of fluorescence in the
former.
Observations were performed with a Nikon Eclipse
E600 Microscope with Nikon Y-Fl epifluorescence
attachment housing a 100 W mercury bulb. UV illumina-
tion was achieved with a UV-1A filter set consisting of
exciter filter (EX 365), dichroic mirror (DM 400) and
barrier filter (BA 400).
Categorization of root zones
The three zones, as defined by McKenzie and Peterson
(1995a,b), were distinguished by visual inspection. The
transition from white to CT zone was marked by a change
from white to brown, which was visible macroscopically.
Inspection of representative cross sections confirmed that
this transition corresponded to gradual death and sloughing
of cortical cells. The change from CT to cork zone was
delineated by inspecting freehand cross sections stained
with FY. The cork zone began where there was a complete
ring of brightly fluorescing cork cells just beneath the
endodermis. With some practice, this information, along
with macroscopic observation, was used to separate the CT
zone from the cork zone. The lengths of the white and CT
zones were measured using a ruler, and the diameter of the
white zone was measured using a caliper.
Growth medium effects
Growth conditions
One-year-old seedlings from the Oklahoma State Forest
Regeneration Center were transplanted into three different
media: (1) mist chamber, (2) peat-vermiculite and (3)
694 Trees (2007) 21:693–706
123
loamy sand. The mist chamber (1.2 m long, 0.6 m wide
and 0.9 m deep) had slits in the top through which seedling
roots were inserted up to the root collar. The bottom of the
mist chamber contained a Hoagland solution (depth of
0.1 m) modified for pine (Kumar 2003). An immersion
pump produced a mist spray for 10 s at 5 min intervals.
Roots grew to approximately 400–600 mm during the
experiment; roots that grew into the solution were imme-
diately removed. The plants were maintained at 24C and
illuminated 14 h each day by a 1,000-W high-pressure
sodium lamp at 150–200 lmol m
–2
s
–1
PPFD at plant
level.
Two further groups of seedlings were transplanted (one
seedling per pot) into pots (0.2 m diameter and 0.2 m deep)
containing either peat-vermiculite (1:1 by volume) or
loamy sand (77% sand, 19% silt, and 5% clay) containing
30.5 mg kg
–1
nitrate nitrogen, 27.0 mg kg
–1
plant-avail-
able phosphorus, and 58.0 mg kg
–1
plant-available
potassium. The pots were in a greenhouse where temper-
atures were maintained at 27–32C during the day and 15–
20C at night. Pots were watered 2–3 times each week and
fertilized every 2 weeks with 20-19-18 Peat-Lite Special
1
(The Scotts Company, Marysville, OH, USA). Solar radi-
ation was approximately 50% full sunlight at plant level
(1000 lmol m
–2
s
–1
PPFD at midday). Where possible,
sample roots were taken from the interior of the soil away
from the pot surface.
The two solid growth media were measured for soil
strength (cone index, 12.8 mm diameter cone, FIELD-
SCOUT SC 900 Soil Compaction Meter, Spectrum
Technologies, Inc., Plainfield, IL, USA), bulk density, and
water content. Five pots of each medium were watered to
saturation and allowed to drain for 48 h before
measurement.
Sampling
In each environment 15–25 roots were randomly sampled
over a period of 12 months beginning in March. One or
two roots were taken from a plant, i.e., 8–13 plants were
sampled. Because the mist chamber and greenhouse had
almost constant growth conditions, except for the seasonal
change in the photoperiod in the greenhouse, the produc-
tion of roots was relatively steady.
Observation of root anatomy
Roots were cut into 10 mm segments starting at the distal
end and placed in water in labeled containers. The number
of cortical cell layers (including the endodermis and dead
outer cells) was counted from the periphery of unstained
sections, as the roots were without a well-defined epidermis
(Mirov 1967). Two counts on opposite sides of each sec-
tion were averaged. The development of the endodermis
was determined by staining for Casparian bands and sub-
erin lamellae and the development of cork cells was
determined by staining, as described above. The numbers
of suberized and non-suberized cells in the endodermis
were counted.
Functionally mature xylem tracheids in the tips of white
roots were detected with Cellufluor dye as described above.
The distal end of the stained tracheids was marked and the
distance from the root apex to this point was measured
using a ruler. The number of xylem strands in each sample
was also counted.
Development and seasonal effects
Growth conditions
Pinus taeda seeds were planted at the Oklahoma State
Forest Regeneration Center on May 2, 2001 in 1.5 by
190 m seedling beds in 7 parallel rows at a spacing of
190 m
–2
. The beds had been sterilized with methyl bromide
1 year earlier and treble super phosphate (100 kg ha
–1
of
P) added 30 days before planting. The seedbeds were
irrigated and fertilized according to operational procedures
to produce high quality seedlings. Herbicides and fungi-
cides were used to control pests and soil pH was
maintained between 5.5 and 6 by addition of mineral
sulfur.
Three replications were established at least 50 m apart
in different beds. Starting on June 11 until December 15,
2001, seedlings were harvested every 30–40 days. Stems
were cut at the soil surface and the shoots measured for
stem heights, stem diameters 1 cm above the ground, and
stem and needle dry weights (measured after 48 h in a
forced-air oven at 70C). A 0.1 m diameter PVC pipe was
used to extract a soil core to a depth of 0.6 m. Four soil
cores were taken randomly from each replicate, two
within and two between rows. The within-row samples
contained four to five seedling shoots and attached root
systems, while the between-row samples contained only
roots; in June there were no roots in the between-row
samples. Soil cores were transported intact to a laboratory
where roots were carefully extracted from each 0.1 m
segment and stored in water at 4C until they were
analyzed.
On October 16 seedling roots were undercut 0.2 m
below the soil surface, and on October 23 lateral roots were
pruned midway between the rows in accordance with
standard operational nursery procedures for P. taeda. Root
pieces cut off by these procedures died, as evidenced by
Trees (2007) 21:693–706 695
123
their dark color and flaccidity. The severed and dead roots
were separated and discarded from the two harvests fol-
lowing root pruning.
Root analysis
Roots were separated into three categories: taproots, lateral
roots and mycorrhizal tips. Images produced by scanning
tap and lateral roots were measured for lengths and diam-
eters of white, CT and cork zones with Delta-T scan
software. White roots were stained with methyl violet (3%
aqueous solution) before scanning. Roots were dried at
70C for 48 h and weighed.
Mycorrhizal tips were handled differently. A mycor-
rhizal tip was characterized as a single, dichotomously
branched root, no longer than 4 mm. A sub-sample of 25
mycorrhizal tips was taken from each core and measured
for diameter and length. Surface area of a mycorrhizal tip
was calculated from the diameter and total length of its
base and branches using the equation for the surface area of
a cylinder. All mycorrhizal tips, including the sub-sample,
were dried at 70C for 48 h and weighed. These data were
used to calculate the total number, length and surface area
of mycorrhizal tips.
Cortical plasmalemma surface area
At each harvest, cortical plasmalemma surface area
(CPSA) was calculated following the method of Taylor and
Peterson (2000). Three cross sections each from the mid-
point of white zone, CT zone and mycorrhizal root from
each within-row sample were used to determine the aver-
age numbers and diameters of cortex cells, and numbers
and tangential widths of passage cells (Figs. 8,9,11).
There is no persistent epidermis in pine roots as it is
sloughed off during early stages of root development
(Mirov 1967). The number of cortex cells was determined
from images of cross sections captured by imaging soft-
ware (Optronics MagnaFire) attached to the microscope
and projected on paper. Images were split into eight sectors
and all cells outside the endodermis were counted in four
sectors opposite each other. The total number of cortex
cells was calculated as twice the counted number. The
diameters of cortex cells and outer tangential widths of
passage cells were measured using Image-Pro Plus (version
4.0).
To determine the average lengths of cortex and passage
cells, three root segments each were removed from the
mid-point of the white (10 mm long), CT (10 to 20 mm
long) and mycorrhizal (entire mycorrhizal root) zones
obtained from each within-row sample. The lengths of
cortex and passage cells were measured from cleared tis-
sues stained with FY as described above (Fig. 13). The
cells to be measured were selected at random: eight to ten
for the white and CT zones and four to six for mycorrhizal
roots. Total CPSA included membrane area in the tan-
gential, radial and transverse surfaces of cortex cells and in
the outer tangential surface of passage cells in the endo-
dermis. Values were expressed per millimeter length of
root.
Cortex cells. The radial and tangential plasmalemma
surface area of cortical cells in a 1 mm segment of root, S
1
(mm
2
), was calculated assuming the cells to be aligned as
cylinders 1 mm long, and ignoring the transverse walls
between adjacent cells according to:
S1¼nh2prc
ðÞ;ð1Þ
where h= 1 mm, r
c
= average radius of cortex cells in
cross section (mm) and n= the number of cortex cells in
cross section. The surface area of the transverse
membranes at the end of each cell in a 1 mm segment of
root, S
2
(mm
2
), was calculated as:
S2¼npr2
c
2hh1
c;ð2Þ
where h
c
= average length of cortical cells (mm) and
2 accounts for the membranes at the two ends of each
cell.
Passage cells. The CPSA of passage cells in a 1 mm
segment of root, P(mm
2
) was determined assuming the
cells were rectangular boxes according to:
P¼npwph;ð3Þ
where n
p
= number of passage cells in cross section,
w
p
= width of outer tangential face and h= 1 mm.
The total CPSA in a 1 mm segment of root (CPSA
pul
,
mm
2
) was calculated as:
CPSApul ¼S1þS2þP:ð4Þ
Means and standard deviations were calculated based on
three replicates. Each replicate was the mean of four
samples, two within and two between seedling rows.
Data analysis
Statistical analyses were performed using SAS, and
means and standard deviations were calculated (SAS
1999). The significance of differences among environ-
ments was tested by the means separation test (LSD,
P£0.05).
Results were plotted against date to show seasonal
trends.
696 Trees (2007) 21:693–706
123
Results
Growth medium effects
Loamy sand compared to peat-vermiculite offered the
greatest resistance to root penetration and least capacity to
hold water (Table 1). At the other extreme, the mist
chamber presented no resistance to penetration and had a
consistent unlimited supply of water.
Pinus taeda seedling roots grown in all three conditions
had a distal white zone easily distinguished by its colour.
Cross sections had a well defined, smooth circular perim-
eter, a feature related to its apparently healthy cortex with
relatively large, nearly round cells (Fig. 1). The innermost
layer of the cortex, the endodermis, developed Casparian
bands 5 mm from the apex (data not shown). Suberin
lamellae (Fig. 2) were numerous at 5 mm from the apex in
roots from solid media and at 15 mm in roots grown in the
mist chamber. Proximal to the white zone was the brown
CT zone that had a cortex with reduced integrity. Collapse
of the cortical cells led to development of a wavy irregular
perimeter, especially in roots from loamy sand (Fig. 3).
This feature became more pronounced with age (distance
from the root apex). The number of non-suberized passage
cells in the endodermis declined with distance from the
apex, and mature xylem tracheids became more numerous.
The cork zone, located proximal to the CT zone, initially
had a ring of cork just below a partially crushed endoder-
mis (Fig. 4), some remnants of cortex, and a very ragged
periphery. Abundant secondary xylem and phloem had
developed in the stele.
A few root features were unaffected by the growing
conditions. The root diameter in the white zone and num-
ber of endodermis cells remained constant (Tables 2,3,
Fig. 14a–c). Conducting xylem extended to within 7–9 mm
of the apex (Fig. 5, Table 2), and non-functional tracheids
with spiral thickenings extended farther toward the root tip.
Two xylem poles were usually present but occasionally 3
or 4 poles were found (Figs. 6[diarch] and 7[triarch]).
A number of root features were influenced by the growth
media. The lengths of the white and CT zones were both
substantially reduced and second-order lateral roots
emerged nearer the apices when roots were grown in solid
media compared with mist culture (Table 2). Results
showed the overall average number of cortex cell layers in
the white zone was greater in the peat-vermiculite medium
than the other two root growth media (P£0.05, Table 3)
which had the same number of layers. The number of cortex
cell layers in the white zone decreased rapidly basipetally
due to death and sloughing (Fig. 14d). The decrease began
sooner and was more rapid in the mist and loamy sand
media. Eventually, only one cortex cell layer remained in
the loamy sand and four to eight in the other two media.
The number of passage cells decreased as the root
growth conditions increased in rigor (Table 3). At the first
appearance of suberin lamellae in serial cross-sections
taken from mist chamber roots (15 mm from the apex),
58% of the endodermal cells were passage cells (Fig. 14a).
In contrast, only 21% of the peat-vermiculite (Fig. 14b)
and 9% of the loamy sand (Fig. 14c) remained passage
cells at the first appearance of suberin lamellae 5 mm from
the root tips. Suberization was complete at 105, 145, and
195 mm from the apex in the loamy sand, peat-vermiculite,
and mist, respectively (Fig. 14a–c). At this point, a unise-
riate layer of cork cells that also stained brightly with FY
had differentiated just internal to the ring of endodermal
cells (Fig. 4).
Development and seasonal effects in nursery-cultivated
seedlings
Seedling growth and morphology
Dry weights of roots, stems, and needles showed slow
growth in June followed by more rapid growth in July and
later (Fig. 15a). The most rapid increase in stems and
needles was in September and October, followed by an
abrupt slowdown in November. In contrast, the most rapid
increase in root dry weight was after undercutting in mid-
October (Fig. 15a). The prolongation of root growth was
associated with temperatures slightly above normal (Fig.
15a). The white, CT, and cork zones (Figs. 8,9,10) were
clearly recognizable. Mycorrhizal roots (Figs. 11,12) first
appeared in August and were abundant by December (Fig.
15c). The final mean shoot height was 251 mm, and the
root collar diameter was 4.7 mm.
Taproots reached 0.3 m soil depth in June and by July a
few had grown to 0.5 m (Fig. 15b). Root growth below
0.2 m was minimal in the hot period of July and August.
Undercutting below 0.2 m in mid-October detached no
more than 30% of the root surface area (Fig. 15b). Despite
undercutting and lateral pruning, the dry weight of roots
increased more than 50% from October to December (Fig.
15a).
Table 1 Soil strength (cone index), bulk density and water content of
root growth media two days after watering (n= 5, mean and standard
deviation)
Characteristic Peat-vermiculite Loamy sand
Cone index (kPa) 0 (0.00) 393 (38)
Bulk density (Mg m
–3
) 0.12 (0.01) 1.66 (0.04)
Water content (m
3
m
–3
) 0.48 (0.03) 0.19 (0.01)
Trees (2007) 21:693–706 697
123
The number of white root tips increased rapidly to
around 6,000 m
–2
(soil surface basis) in early July (Fig.
15c), and then more slowly to 9,000 m
–2
by October and
13,000 m
–2
in December. Mycorrhizal tips (Fig. 15c) did
not appear until August and their numbers increased
abruptly after October to over 60,000 m
–2
in December.
The mycorrhizal roots and plentiful fruiting bodies
resembled those of Thelephora terrestris Pers. ex Fr
(Castellano and Molina 1989), the most common mycor-
rhizal fungal species in bare-root and container seedling
nurseries in the United States.
The total root surface area (white, CT, cork, and
mycorrhiza) increased from 0.6 m
2
m
–2
in early June to
7.9 m
2
m
–2
in mid-December (Fig. 15d). The drop in root
surface area in October due to root pruning was followed
by a very rapid increase in December.
Surface areas of individual root zones
The white zone grew early to a specific length where it
remained throughout the experiment. Consequently its
percentage of total root surface area decreased from 25% in
July to just below 5% in December (Fig. 15d). Mycorrhizae
did not appear until August and never amounted to more
than 3% of the total surface area. The surface area of the
CT zone increased 14 fold from June to December and
always contributed over 70% of the total. The cork zone
Figs. 1–7 Photographs were of P. taeda root cross sections except 5,
which was a squashed segment. The root preparations were stained
with fluorol yellow 088 unless otherwise specified. All sections were
viewed with UV light. 1Mist chamber white zone at 5 mm from tip
stained with berberine-aniline blue. A multilayered cortex encloses
the central stele; the endodermis was not suberized at this region. 2
Mist chamber white zone at 30 mm in from apex containing over 15
passage cells. 3Loamy sand condensed tannin (CT) zone at 70 mm
from apex. Death of outer cortex cells began centripetally following
suberization of endodermis. Triarch xylem poles grew centripetally to
merge at the center. 4Loamy sand cork zone at 150 mm from the
apex. A cork layer initiated internal to endodermis crushing it and
xylem poles merged at the center of stele. 5Conductive portion of a
diarch xylem in the white zone. The xylem was loaded with cellufluor
(see text for details). The arrow indicates direction of stain movement
through tracheids and arrowhead indicates water flow through intact
root. 6Peat-vermiculite CT zone at 130 mm from apex. Death of
cortex progressed from periphery and most endodermal cells were
suberized leaving only 1 or 2 passage cells. 7Loamy sand CT zone at
110 mm from apex. Death of cortex progressed leaving only 1 or 2
layers of cells and most endodermal cells were suberized. Bar
100 lm. cCortex, en endodermis, kcork layer, pphloem, pc passage
cells, pe pericycle, rresin duct, sstele, ttracheids, xxylem
698 Trees (2007) 21:693–706
123
surface area increased to nearly 25% of the total in October
and then declined slightly after root pruning.
CPSA
Each root zone differed in its contribution to the overall
root CPSA (CPSA
tot
) due to differences in length and
CPSA per unit root length (CPSA
pul
; Table 5). The white
zone always contributed most of the CPSA
tot
(Fig. 15e),
i.e., over 95% in June through August and 65% in
December when mycorrhizal roots increased to nearly 35%
of CPSA
tot
. The CT zone contribution was small, usually
below 5%, and the cork zone contributed nil.
Large increases in white zone cortical cell numbers and
surface area from June to December (Tables 4,5) led to a
nearly threefold increase in CPSA
pul
. Similarly, the three-
fold increase in mycorrhizal root CPSA
pul
from August to
December (Table 5) was due to a 50% increase in cortex
cell number and diameter (Table 4).
Among the 44–57 endodermal cells in the white and CT
zones, only passage cells had CPSA accessible to the
modified soil solution (MSS). These cells became more
important in the CT zone owing to the death of the
remainder of cortex. In the white zone about 20% of
endodermal cells were passage cells regardless of the time
of year, whereas the passage cells of the CT zone decreased
from 11 to 4% from June to December (Table 6). The
contribution of passage cells to CPSA
pul
was less than 2%
in the white zone and 100% in the CT zone (Table 5).
Although 21–24% of the 11–13 endodermal cells were
passage cells in a mycorrhizal root, they never contributed
more than 1% of the CPSA
pul
.
Discussion
A major finding of this study was that P. taeda responded
to growth media with contrasting water supply and pene-
tration resistance by producing different kinds of roots.
Under stressful conditions the white and CT zones were
shorter and more suberized. As a possible counter to the
reduction in these zones there was a concomitant acceler-
ation in production of second-order laterals. Another major
finding was the capacity for very rapid production of
mycorrhizae and recovery following undercutting and root
pruning. Although the white zone and mycorrhizae con-
tributed only a very small portion of root length, they
contributed the dominant portion of the CPSA
tot
in part due
to the capacity to increase threefold their CPSA
pul
.
The effects of growth conditions on root development
in the laboratory
Features not influenced by growth conditions
The lack of an environmental effect on root diameter of the
white zone has important implications for root function
because root diameter determines the root surface area
through which water and ions pass, and the length of the
radial pathway from the soil solution to the xylem
(Landsberg and Fowkes 1978; Sperry et al. 1998; Rieger
and Litvin 1999). The results also showed the hydraulically
isolated tip, from which water and ions cannot be readily
Table 2 Morphological
characteristics of white and
condensed tannin (CT) zones in
the first-order lateral roots from
mist chamber, peat-vermiculite,
and loamy sand
Means within rows followed by
different letters are different at
P£0.05, n= 10–20
Root trait Mist chamber Peat-vermiculite Loamy sand
Diameter of white zone (mm) 0.74 0.68 0.73
Length of white zone (mm) 47.93
a
25.50
b
29.33
ab
Length of CT zone (mm) 152.23
a
124.67
b
80.83
c
Distance from apex
First conducting xylem (mm) 6.63 8.91 8.53
Emergence of 2lateral (mm) 65.00
a
55.74
a
28.10
b
Beginning of cork zone (mm) 200.00
a
150.00
b
110.00
c
Table 3 Number of cells in root cross sections of white and con-
densed tannin (CT) zones in the first-order lateral roots from mist
chamber, peat-vermiculite, and loamy sand
Root zone and
cell type
Mist
chamber
Peat-vermiculite Loamy
sand
White zone
Cortex cell layers (no.) 7
b
10
a
7
b
Endodermal cells (no.) 69 62 65
Passage cells (no.) 33
a
11
b
5
c
Passage cells (%) 48 18 8
CT zone
Cortex cell layers (no.) 4
b
8
a
1
c
Endodermal cells (no.) 70 62 66
Passage cells (no.) 19
a
5
b
2
c
Passage cells (%) 28 8 3
Means within rows followed by different letters are different at
P£0.05, n= 9–22
Trees (2007) 21:693–706 699
123
transported to the shoot, occupied a short and fairly con-
stant length at the tip of the white zone. Enstone et al.
(2001) found a similar value for pouch- and pot-grown
seedlings (2–8 mm). These findings may simplify and
support assumptions made when modeling water move-
ment through roots (Landsberg and Fowkes 1978; Sperry
et al. 1998).
The results concerning the number of xylem poles and
number of endodermal cells indicated that the patterning of
these tissues and the number of cell divisions leading to their
production in the root apex were unaffected by the imposed
growth conditions. Plant age may be factor in number of
endodermal cells, as second-year seedlings had 30% more
(62–70, Table 3and Fig. 14a–c) than first-year seedlings
(44–57, Table 6). The only other study of this type in loblolly
pine found 40–45 endodermal cells in tap roots of first-year,
pouch- and pot-grown seedlings (Enstone et al. 2001).
Features affected by growth conditions
Mist culture with the least mechanical and water stress
produced roots characteristic of rapid growth: long white
and CT zones (Table 2), reduced suberization, reduced
cortical cell sloughing (Fig. 14d) and lateral root emer-
gence at greater distance from the root tip. The less
favorable growing conditions in solid media caused
changes in the root that would have reduced the per-
meability of the roots to water, unless compensated for
by increased numbers of aquaporins (Barrowclough et al.
2000), and would have allowed them to respond to
drought conditions quickly. The reduction in the length
of the white zone, presumed major site of ion uptake due
to its large CPSA, was countered to some extent by
production of second-order lateral roots nearer the apex
and in peat-vermiculite culture by the increased number
of layers of cells in the central cortex. More taxing
stresses, such as those encountered in nature, could be
expected to have even more striking effects on root
development. An earlier study found Quercus alba roots
decreased elongation rate and increased, the number of
apices as soil water potential decreased (Teskey and
Hinckley 1981). This was interpreted as an efficient
strategy for increasing root surface area and in part
responsible for Q. alba’s capacity to thrive on droughty
sites.
Figs. 8–13 Anatomical analysis of root zones of first-year nursery
grown P. taeda seedlings. Figures 8–11 show cross sections stained
with fluorol yellow 088. Figures 12 and 13 are cleared whole mounts
stained with fluorol yellow 088 (Fig. 13) or Chlorazol black E
(Brundrett and Kendrick 1988; Brundrett et al. 1984) (Fig. 12). 8
White zone 20 mm from the apex. The central stele was surrounded
by a multilayered cortex limited by a single layered endodermis
containing passage cells. The section was divided into 8 sectors for
counting cortex and passage cells and measuring the diameter of
cortex cells and outer tangential width of passage cells. 9CT zone
120 mm from the apex. Death of cortex cells had progressed from the
periphery although some of them were retained. The passage cells
were fewer than in the white zone. The diarch xylem had matured
centripetally toward the stele center. 10 Cork zone 180 mm from the
apex. The endodermis was completely suberized and a cork layer
initiated within the pericycle expanded to crush the endodermis. 11
Mycorrhizal root. The intercellular spaces among cortex cells were
filled with fungal hyphae. The number of cortex cells and endodermal
cells were less than in the white zone of a non-mycorrhizal root. 12
The tip of a mycorrhizal fork cleared and stained with Chlorazol black
E. 13 Tip of a non-mycorrhizal root cleared using 10% bleach and
stained with fluorol yellow 088. Length of cortex and passage cells
were measured from the cleared and stained roots to calculate cortical
plasmalemma surface area. Bar 100 lm. Abbreviations: ccortex, en
endodermis, kcork layer, mmantle, pphloem, pc passage cells, pe
pericycle, rresin duct, sstele, and xxylem
700 Trees (2007) 21:693–706
123
Root development under nursery conditions
It is interesting to note that the total root surface area in the
nursery at the end of the first growth period was compa-
rable to that of much older forest-grown trees (8 vs.
2–15 m
2
m
–2
for trees 10–35 years old; Kramer and Bull-
ock 1966; McCrady and Comerford 1998; Hacke et al.
2000).
White zone
The small percentage of white zone (\5%) at the end of the
first year is consistent with earlier field studies where white
roots of much older trees constituted a small part of the root
system (Reed 1939; Kramer and Bullock 1966; Wilcox
1968; McCrady and Comerford 1998). The amount of
white zone was determined by the number of white root
tips, their rate of elongation, and the rate of their matura-
tion to CT zones or mycorrhizal roots. The number of P.
taeda white root tips more than doubled from July to
December (Fig. 15c), but the white zone surface area did
not increase (Fig. 15d). Apparently, the length of white
zone per root tip declined due to accelerated maturation to
CT zone and development into mycorrhizal roots.
Mycorrhizae
Although mycorrhizae were not produced until after July,
due to very rapid growth they attained about 2.8% of the
total root area in December (Fig. 15d), close to values for
older stands (2.7–5.3%, Kramer and Bullock 1966;
McCrady and Comerford 1998). The relatively late pro-
duction of mycorrhizae was probably a result of sterilizing
the soil with methyl bromide the fall prior to planting. In
addition, the application of abundant fertilizer and irriga-
tion water may have reduced the development of
mycorrhizal roots (Maronek et al. 1982; Marx et al. 1982;
Ekwebelam and Reid 1983; Danielson et al. 1984).
Mycorrhizae have long been regarded as an adaptation to
increase water and nutrient uptake especially in infertile
soils (Ashford et al. 1988; Smith and Read 1997). The
rapid production of mycorrhizae may be a very beneficial
strategy to increase root uptake capacity rapidly when
needed rather than continuously maintaining an extensive
and costly root system.
Fig. 14 Effects of root growth medium on endodermal suberization
and death and sloughing of cortex cells in the white and condensed
tannin (CT) zones in the first-order lateral roots of Pinus taeda.
a–c Number of non-suberized and suberized endodermal cells. The
arrow indicates transition from white to CT zone. aMist chamber
roots had Casparian bands but no suberin lamellae in their endodermis
in the 0–10 mm segment. Although Casparian bands had formed,
there were no suberin lamellae. bIn peat-vermiculite, suberization
was more advanced closer to the apex compared to mist chamber
roots. cLoamy sand roots had the most advanced suberization close to
the apex. dNumber of layers of cortex cells in the first-order lateral
roots. Thin lines indicate white zone and thick lines indicate CT zone.
Cortex cell layers were retained in the CT zone even after the cell
died (n= 9–22)
0
10
20
30
40
50
60
70
a
sllecl
amredodneforebmuN
0
10
20
30
40
50
60
70
b
0
10
20
30
40
50
60
70
c
Non-suberized
Suberized
Distance from root apex (mm)
0 20 40 60 80 100 120 140 160 180 200 220
sreyalxet
roc
forebmuN
0
2
4
6
8
10
12
Mist
chamber
Loamy
sand
Peat-
vermiculite
A
d
b
Trees (2007) 21:693–706 701
123
CT zone
Because production of the CT zones kept pace with the
overall growth of the root system it always accounted for
over 70% of total surface area (Fig. 15d). Similar results
were obtained in P. banksiana seedling roots growing
outdoors in sandy soil in northern Ontario (Taylor and
Peterson 2000). This zone was not clearly distinguished
from the cork zone until the study of McKenzie and Pet-
erson (1995a), yet it constituted the majority of the root
mg(thgiewyrD 2- )
0
200
400
600
800
1000
1200
1400
0
5
10
15
20
25
30
35
(
erutarepmeT o)C
Air temperature
Normal temperature
Stem weight
Needle weight
Root weight
mrebmun(s
p
ittoorfor
ebm
unev
i
t
a
lumu
C2- )
0
20000
40000
60000
80000
Mycorrhizae
White roots
Prune
lateral
rootss
Undercut
taproot
m(aeraecafrustoorev
it
al
u
muC 2m2- )
0
2
4
6
8
10
Cork zone
CT zone
Mycorrhizae
White zone
Undercut
taproot
Prune
lateral
roots
White zone
m(
ASP
Cev
italum
uC 2m2- )
0
2
4
6
8
10
White zone
CT zone
Mycorrhizae
Jun Jul Aug Sep Oct Nov Dec Jan
Harvest Date
Jun Jul Aug Sep Oct Nov Dec Jan
Harvest Date
a
m(aeraecafrustoorevitalumuC 2m2
-)
c
d
e
b
0
2
4
6
8
0-10 cm
10-20 cm
20-30 cm
30-40 cm
40-50 cm
Fig. 15 Seasonal changes in the roots of first-year nursery-grown
loblolly pine (Pinus taeda) seedlings. Taproots were undercut below
20 cm and laterals were pruned in October. aDry weights of stems,
needles, and roots. Weather parameters included mean monthly air
temperature at Washington, OK (Mesonet 2001) and normal temper-
ature (30-year average) at Purcell, OK (NOAA 2001). bDistribution
of root surface area by depth. cCumulative numbers of white root tips
and mycorrhizae tips. dCumulative surface areas of white, condensed
tannin (CT), cork, and mycorrhizal root zones. eCumulative CPSA in
the white, CT, and mycorrhizal root zones. Cumulative values shown
on the graphs for a date are the sums of the means for different types
of root tips and zones. n= 3 and bar = ± standard error
702 Trees (2007) 21:693–706
123
system of these species. Although the CT zone had only a
small fraction of its surface area non-suberized and capable
of ion uptake, the great length of this root zone meant that
it was exposed to a large volume of soil. The benefit of this
dispersed uptake capacity is exploring widely in soils
where the availability of water and nutrients can be highly
heterogeneous; it would increase the likelihood of part of
the root system having contact with available mineral
nutrients and water.
Cork zone
The cork zone was the oldest part of the root system and
occupied 20–25% of the total root surface area after July
(Fig. 15d). Its abundant secondary xylem and continuous
cork layer (Fig. 10) made it an excellent conduit for
transporting water from distal root zones to the shoot.
Earlier studies showed the cork layers of P. banksiana were
capable of blocking the movement of berberine, although
the presence of a Casparian band-like structure was not
proven (McKenzie and Peterson 1995b). It was suggested
that if the cork zone of the root had the same properties as
stem cork, at maturation it should present high resistance
apoplastic and cell-to-cell pathways (Peterson et al. 1999).
CPSA
The proportion of the root length occupied by the various
root zones provided an overview of root structure but is
only part of the picture. The developmental pattern of tree
roots with their three anatomically distinct zones strongly
suggested the possibility of different absorptive functions
along individual roots. Each zone was very different from
the others in the following traits: (1) amount of CPSA, (2)
amount of suberin lamellae in the endodermis and (3)
presence of cork cells. Assuming that the ions in the MSS
do not diffuse through Casparian bands of the endodermis
or the cork cells of the periderm to a significant extent, the
Table 4 Seasonal changes in
cortex cell number, diameter,
and length in the white zone and
mycorrhizae from June until
December
Mycorrhizae did not appear
until August. Mean and
(standard error), n=3
Root zone and harvest month Cortex cells
Number Diameter (mm) Length (mm)
White zone
June 102.2 (0.347) 0.035 (0.002) 0.174 (0.010)
July 143.1 (0.929) 0.041 (0.001) 0.166 (0.017)
August 167.7 (1.114) 0.038 (0.003) 0.169 (0.001)
October 238.7 (3.656) 0.038 (0.002) 0.174 (0.005)
November 218.2 (4.726) 0.043 (0.004) 0.144 (0.004)
December 211.7 (2.737) 0.045 (0.003) 0.126 (0.001)
Mycorrhizae
August 50.5 (0.644) 0.032 (0.005) 0.057 (0.001)
October 59.6 (1.337) 0.042 (0.002) 0.066 (0.005)
November 65.0 (0.658) 0.044 (0.003) 0.070 (0.004)
December 76.1 (1.240) 0.058 (0.001) 0.074 (0.004)
Table 5 Cortical plasmalemma surface area per mm of root length
(CPSA
pul
) in the white zone, condensed tannin (CT) zone, and
mycorrhizae
Root zone and harvest month S
1
S
2
P CPSA
pul
White zone
Jun 11.261 1.135 0.200 12.596 (0.967)
Jul 18.563 2.311 0.340 21.213 (0.627)
Aug 19.747 2.193 0.277 22.217 (1.956)
Oct 28.407 3.092 0.277 31.776 (1.534)
Nov 29.185 4.308 0.216 33.709 (3.914)
Dec 29.917 5.364 0.312 35.593 (2.854)
CT zone
Jun – – 0.129 0.129 (0.009)
Jul – – 0.226 0.226 (0.006)
Aug – – 0.137 0.137 (0.008)
Oct – – 0.176 0.176 (0.004)
Nov – – 0.093 0.093 (0.004)
Dec – – 0.048 0.048 (0.005)
Mycorrhizae
Aug 5.138 1.460 0.063 6.661 (1.138)
Oct 7.826 2.471 0.073 10.369 (0.912)
Nov 8.98 2.810 0.090 11.881 (0.825)
Dec 13.856 5.408 0.096 19.361 (0.454)
Mycorrhizae did not appear until August. S
1
tangential and radial
surfaces of cortex cells, S
2
transverse surface of cortex cells, Pouter
tangential surface of passage cells; mean and (standard error),
CPSA
pul
=S
1
+S
2
+P,n=3
Values are in mm
2
mm
–1
Trees (2007) 21:693–706 703
123
relevant surface area for ion absorption resides in the
plasmalemma of the living cortical cells. The CPSA is one
feature that can be quantified by an anatomical investiga-
tion and be expected to influence the uptake of water and
ions by roots. On one hand, it is the location of carrier
proteins for the various ions, allowing them entry into the
symplast and ultimately into the root stele (see Smith 2002;
Epstein and Bloom 2005). On the other hand, it provides a
resistance to water flow on its radial path from the soil
solution into the root xylem (see Peterson and Steudle
1993); the extent of this resistance is controlled by the
activity of aquaporins in the membranes (see Ye and
Steudle 2006).
The greatest part of the CPSA
tot
in roots of nursery-
grown P. taeda seedlings was always in the white zone
because it retained a living cortex. Although white zone
length did not vary, its CPSA
pul
varied by a factor of three;
therefore, the white zone may vary tremendously in ion
uptake capacity due to changes in cell number and surface
area and activities of the carrier proteins (Smith 2002;
Epstein and Bloom 2005). The white zones should be
costly to maintain, and it may only be effective for the
plant to produce small lengths. Production of white zone
with high CPSA
pul
can increase when the roots are
wounded by pruning to replace lost ion uptake capacity
quickly, when roots grow into soil rich in ions and when
plants have a high demand for mineral nutrients.
Mycorrhizal roots also had a live cortex and contributed
a significant part of CPSA
tot
after August (Table 5, Fig.
15e) reaching 35% of CPSA
tot
in December. This was less
than the 75% contribution recorded for mycorrhizae in P.
banksiana nursery seedlings (Taylor and Peterson 2000),
but in their study, the soil had been amended with an in-
noculum of mycorrhizal fungus. Whether or not the cortical
cells of mycorrhizal roots take up ions directly from the
MSS depends on the permeability of the fungal mantle that
encloses them (Ashford et al. 1988; Behrmann and Heyser
1992; Vesk et al. 2000;Bu
¨cking et al. 2002). This, in turn,
depends on the age of the association, and on the fungus
involved (Ashford et al. 1988; Peterson et al. 2004). Ex-
tramatrical hyphae transport ions to the Hartig net in the
intercellular spaces of the cortex (Marshner and Dell 1994;
Peterson and Bonfante 1994) where they can be transferred
to the plant (Peterson and Massicotte 2004). Thus, the ion
uptake capacity of a mycorrhizal root can be substantially
larger than predicted by the surface area of its cortical cells.
Despite its major contribution to the length of the
seedling roots, CPSA
tot
of the CT zone was very small
(only 2–9% of the total). Only a small membrane surface
was available for ion uptake due to death of the cortical
cells and the production of suberin lamellae in the majority
of the endodermal cells. Thus, the only membrane avail-
able for ion uptake was the one lining the outer tangential
walls of the endodermal passage cells. The passage cells
could allow some water and ion absorption from moist
soils, and the extensive suberization of the endodermis
would protect the enclosed part of the root from water loss
in dry soils.
Table 6 Seasonal changes in
number of endodermal cells and
number, tangential width, and
length of passage cells in the
white zone, condensed tannin
(CT) zone, and mycorrhizae
from June until December
Mycorrhizae did not appear
until August. Mean and
(standard error), n=3
Root zone and harvest month Endodermal cells Passage cells
Number Number Width (mm) Length (mm)
White zone
June 43.7 (0.551) 8.0 (1.079) 0.025 (0.0004) 0.116 (0.004)
July 48.7 (0.709) 13.3 (0.825) 0.026 (0.0005) 0.116 (0.005)
August 49.0 (0.709) 11.0 (1.248) 0.025 (0.0020) 0.160 (0.012)
October 56.3 (1.665) 12.1 (1.082) 0.023 (0.0020) 0.122 (0.003)
November 54.6 (1.652) 9.3 (0.502) 0.023 (0.0010) 0.105 (0.004)
December 57.2 (1.960) 11.1 (1.634) 0.028 (0.0010) 0.103 (0.003)
CT zone
June 48.3 (0.586) 5.3 (0.297) 0.024 (0.0010) 0.142 (0.011)
July 51.0 (1.464) 9.0 (0.529) 0.025 (0.0010) 0.150 (0.010)
August 53.3 (0.173) 5.2 (0.385) 0.026 (0.0020) 0.143 (0.003)
October 55.1 (1.856) 7.0 (0.191) 0.025 (0.0010) 0.156 (0.008)
November 54.2 (1.253) 3.6 (0.064) 0.026 (0.0010) 0.115 (0.005)
December 53.0 (0.961) 2.0 (0.230) 0.024 (0.0003) 0.121 (0.001)
Mycorrhizae
August 11.3 (0.289) 2.4 (0.082) 0.026 (0.0020) 0.087 (0.008)
October 12.4 (0.404) 2.6 (0.231) 0.028 (0.0010) 0.093 (0.007)
November 12.7 (0.702) 3.0 (0.330) 0.030 (0.0003) 0.088 (0.012)
December 13.4 (0.850) 3.2 (0.294) 0.030 (0.0020) 0.115 (0.010)
704 Trees (2007) 21:693–706
123
The anatomy of the cork zone with its outer layer of
cork cells means that no CPSA is present in this zone.
Seemingly in contrast to this, results reported in several
papers indicate both passive and active uptake of P, N and
K in brown, woody and secondary roots (Crider 1933; Van
Rees and Comerford 1990; Sougnez-Remy et al. 1993; van
Praag et al. 1993; Escamilla and Comerford 2000). How-
ever, as these reports do not include detailed anatomical
observations, it is not possible to know whether the mea-
surements on the brown, woody and secondary roots
included passage cells such as those in the CT zone. In any
case, these findings highlight the need for further ana-
tomical studies of the older cork and CT zones to determine
potential pathways of ion uptake.
The P. taeda root system showed a high level of plas-
ticity among environments and across seasons. It had the
capacity to change the lengths and characteristics of root
zones and to accelerate the production of mycorrhizae and
new roots. The changes in morphology and anatomy
appeared to have adaptive value but this must be confirmed
by further study. Given the large amount of environmental
variation in root traits, another important question that
deserves further study is how much genetic variation does
P. taeda have for these root traits.
Acknowledgments The authors would like to acknowledge the
significant contribution of the late David Ferris. The research was
done with the support and cooperation of the Oklahoma Forest
Regeneration Center. This research was approved for publication by
the Director of the Oklahoma Agricultural Experiment Station and
supported in part by project MS 1979.
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