Journal of Experimental Botany, Vol. 51, No. 345, pp. 755–768, April 2000
Mapping meristem respiration of Prunus persica (L.)
Batsch seedlings: potential respiration of the meristems,
O diffusional constraints and combined effects on root
2
growth
L.P.R. Bidel1, P. Renault2,4, L. Pagès3 and L.M. Rivière1
1 INRA, 42 rue Georges Morel, BP 57, 49071, Beaucouzé, France
2 INRA, Unité de Science du Sol, Domaine St-Paul, Site Agroparc, 84914 Avignon Cedex 9, France
3 Unité d’Ecophysiologie et Horticulture, Domaine Saint-Paul, Site Agroparc, 84914 Avignon Cedex 9, France
Received 25 October 1999; Accepted 4 November 1999
Abstract
Introduction
Root system architecture partially results from meristem activities, which themselves depend on endogenous and environmental factors, such as O depletion.
2
In this study, meristem respiration and growth was
measured in the root systems of three Prunus persica
(L.) Batsch seedlings. The spatial distribution of meristem respiration within the root system was described,
and the relationship between the respiration rates
and meristem radii was analysed, using a model of
radial O diffusion and consumption within the root.
2
Histological observations were also used to help interpret the results. Respiration rates were linearly correlated to the root growth rates (r2=0.9). Respiration
reached values greater than 3.5×10−13 mol O s−1 for
2
active meristems. The taproot meristem consumed
more O than the rest of the entire root system meris2
tems. Similarly, the first order lateral meristems used
more O than the second order ones. A near hyperbolic
2
relationship between respiration rates and meristem
radii was observed. This can be explained by a model
of radial O diffusion and consumption within the root.
2
Therefore, only one maximum potential respiration
rate and one O diffusion coefficient was estimated
2
for all the meristems.
Hierarchical organization of root system architecture (as
described by Atger and Edelin, 1994), results from meristematic activity, cell elongation, differentiation processes
along axes, and the initiation and development of laterals
of various orders. The primary meristem plays a major
role in plant development. It is often considered that the
quiescent centre is involved in generating different numbers of cell ranks and numbers of vascular strands
( Torrey, 1957; Feldman and Torrey, 1975; Torrey and
Feldman, 1977; Rost and Jones, 1988). It determines
partly the vascularization of the primary structure, which
determines nutrient transfer capacities for the axis ( Eshel
and Waisel, 1996). Therefore, understanding root system
formation requires a more precise knowledge of meristem activity.
A number of factors affect meristematic activity, such
as competition for carbon assimilates (Gersani and Sachs,
1992; Bingham and Stevenson, 1993), and hormonal
relations ( Torrey and Feldman, 1977; Torrey, 1986;
Wightman and Thimann, 1980; Wightman et al., 1980).
The more strongly the main axis grows, the more it
apparently inhibits its laterals (Atzmon et al., 1994a, b).
Lateral roots have smaller meristems than a main root
axis, while increasing orders of lateral roots have progressively smaller meristems (Cahn et al., 1989; Varney et al.,
1991; Varney and McCully, 1991). Changes in the apical
diameter, which reflects the size of the meristem, have
been linked to the root growth rate ( Wilcox, 1962, 1968;
Hackett, 1969; Cahn et al., 1989; Pagès, 1995; Thaler and
Key words: O -microelectrode, meristem respiration,
2
spatial distribution, root system architecture, Prunus
persica (L.) Batsch.
4 To whom correspondence should be addressed. Fax: +33 4 32 72 22 12. E-mail: [email protected]
© Oxford University Press 2000
756 Bidel et al.
Pagès, 1996a). It was shown that the meristem diameter
can vary according to the carbohydrate supply (Pagès,
1995; Thaler and Pagès, 1996a). The size of the meristem
may also vary during its period of activity, leading to
axes of varying behaviour (Pagès, 1995; Thaler and Pagès,
1996a, b). However, it was reported that the apical
diameter of lateral roots of oak was not closely correlated
with their growth rate but was still indicative of their
potential growth rate (Pagès, 1995).
The effect on root growth of a decrease in meristem
activity caused by hypoxia has received little attention.
However, hypoxia can be common at ambient [O ],
2
especially for large-diameter meristems (Drew, 1997).
Root apical zones, having high local respiration rates
(R ) and few intercellular air spaces to conduct O ,
root
2
may experience hypoxia in their centre at temperatures
in the range of 298–308 K (Armstrong and Beckett,
1985). In accordance with diffusion-model predictions,
several direct [O ] measurements have indicated hypoxia
2
within metabolically active root tissues, such as maize
primary meristems and elongating segments (Armstrong,
1994; Armstrong et al., 1994; Ober and Sharp, 1994;
Stepniewski et al., 1998). The measurement of other
metabolic indicators, such as alanine, ethanol, lactic acid,
as well as elevated activities of alcohol dehydrogenase
and pyruvate decarboxylase confirmed the occurrence of
anaerobiosis for roots exposed to 21% O (Crawford,
2
1982; Saglio and Pradet, 1980; Saglio et al., 1984; Gibbs
et al., 1995; Crawford and Braendle, 1996). Despite the
role of meristems in morphogenesis, few studies deal with
its respiration and oxygenation, because respiration in
the meristem alone is difficult to measure. Attempts have
been made with sets of equivalent root tips, 3, 5 or 10 mm
long, generally introduced into a stirred nutrient solution
bathing an O -Clark macro-electrode (Saglio et al., 1983;
2
Williams and Farrar, 1992; Brouquisse et al., 1992; James,
1994). This procedure did not distinguish between the
activities of the meristem and the elongation zone
(James, 1994). An analysis of the limiting effects of O
2
on meristem activity has never been conducted on meristems of different sizes and growth potentials representative
of the variability found in the root system. An approach
based on the meristem size-dependent respiration could
be proposed if O diffusion coefficient and specific respira2
tion rate in O non-limiting conditions did not vary
2
between meristems. In this case, meristem bulk respiration
would only depend on size and the [O ] at the root
2
surface. Similarly, meristem activities in the same acropetal sequence or on the whole-root-system architecture
have not yet been compared. This may be of interest for
studying the effect of relative meristem activity on apical
dominance processes.
Because respiration in meristematic tissue is primarily
related to biosynthesis (Amthor, 1989), meristem respiration was studied to test if it was a good indicator of root
growth. Using this meristem activity indicator, an attempt
was made to map meristem respiration throughout the
seedling root system of Prunus persica, in order to check
whether meristem activity depends on morphological and
anatomical criteria, as supported by the competition
theories for C allocation (Bingham et al., 1996; Thaler
and Pagès, 1998). Meristem respiration as a function of
meristem diameter and [O ] at the root surface was
2
analysed in order to determine how much O diffusion
2
within the root tissues can explain differences in
respiration levels.
Materials and methods
Plants and cultivation media
Seeds of Prunus persica L. Batsch GF305 (Nursery Lafond,
Valréas, France) were surface-sterilized in a solution of HClO
(0.5% for 20 min) and washed with continuously oxygenated
deionized water for 2 h. The seeds were then stratified for 3
months in the dark at 277 K in sealed moistened Petri dishes.
After this time, the radicle had reached 5–10 mm in length. The
germinated seeds were rapidly disinfected and washed again
and placed on agar gel.
Plants were grown in agar in order to prevent convective
movements of O , which allowed a diffusion model to be
2
applied to the measurements of O concentration around the
2
root. Although the agar was likely to affect root growth, it
was considered that the advantages of agar outweighed this
disadvantage. Agar gel at a concentration of 6 g l−1 was
dissolved in boiling N/2 Hoagland nutrient solution (Hoagland
and Arnon, 1950) previously mixed with 2 bactericides (nystatin,
1.0 mg l−1; tetracycline hydrochoride, 1.25 mg l−1, SigmaAldrich, Saint Quentin, France) and Rovral fungicide (1.25
g l−1; Rhône-Poulenc, Lyon, France) in order to minimize
microbial proliferation. Acidity was controlled by adding
2.0 mM of MES buffer [2-(N-morpholino)ethanesulphonic acid ]
(Sigma-Aldrich, Saint Quentin, France), following the recommendation which confirmed that this chemical does not disturb
root growth (Ewing and Bobson, 1991). Acidification of the
rhizosphere was monitored with 0.06% bromocresol purple. In
the absence of MES buffer, the colour of agar surrounding the
growing roots became bright yellow within 3 d (indicating a
pH of about 4.0–4.5), instead of taking more than 2–3 weeks
with MES, depending on the roots.
Agar approximately 3–5 mm thick was solidified by cooling the nutrient solution (at 308 K ) in plastic boxes
(20×20×1.5 cm). Germinated seeds were fixed in place with
mastic ( TerostatB) over the gel in which the radicle was settled.
A cellophane film was put on the whole preparation to prevent
microbial contamination and drying. A hole in this film was
made to allow the epicotyl to grow. To prevent the roots from
coiling up, the boxes were slightly inclined (angle from vertical:
10%). The plants were raised in the laboratory at 292–293 K.
Lighting was low (about 150 W m−2), and large quantities of
nutrient solution were poured at daily intervals over the gel
surface. Root growth was recorded by tracing on an acetate
film. The pH became strongly acid about 10–15 d (pH #4)
after cultivation began.
At the meristem level, R
(mol O m−3 tissue s−1) was
root
2
measured on three 15-d-old plants, about 12–15 cm tall with a
stem holding 8–12 fully expanded leaves. Plant no. 1 was used
to check the reliability of R
estimates. Nearly all the
root
Respiration of root meristems 757
meristems on plant no. 2 were analysed for respiration rates.
On plant no. 3, measurements were taken 2–3 times per day
over 4 d, R
being measured both on the taproot meristem
root
and on four early lateral root meristems.
Local estimate of meristem respiration
In order to map R
radial O profiles were performed around
root
2
each meristem, using O -microelectrodes. R
was calculated
2
root
(according to Højberg and Sørensen, 1993; Bidel., 1999):
=−
root
r
2
×D
×
O2−gel
root
A B
∂[O ]
2
∂r
(1)
r=rroot
where R
is the root respiration rate (mol O m−3 tissue s−1),
root
2
r
the root radius (m), D
the diffusion coefficient of O
root
O2−gel
2
the [O ] gradient
in agar gel (m2 s−1) and (∂[O ]/∂r)
2
2
r=rroot
at the root surface (mol m−4). D
was measured by the
O −gel
method of Sierra et al. (Sierra et 2 al., 1995): the value was
similar to that of O diffusion in water. The gradient
2
was estimated by fitting a model of O radial
(∂[O ]/∂r)
2
2
r=rroot
diffusion (Højberg
and Sørensen, 1993) within the gel to [O ]
2
experimental data taken in the 0–500 mm region around the
root surface, using a non-linear fitting procedure (Bard, 1974).
The chosen model accounts for decreasing microbial respiration
within the gel with increasing distance from the root and
estimates local root respiration rates of Prunus better than
other models described in the litterature (Højberg and Sørensen,
1993; Bidel, 1999). In a steady state (i.e. ∂[O ]/∂t=0 where t is
2
time), the model asserts:
R
A
B
1
∂[O ]
k
∂
2 −
0= ×
D
r
O2−gel ∂r
r ∂r
r
A
(2)
where r is the radial position (m), and k is a constant (mol
O m−2 gel s−1) associated with the hyperbolic decrease in
2
microbial respiration k/r.
O -microelectrodes (proposed first by Revsbech and Ward,
2
1983; Revsbech, 1989), were used to take [O ] profiles around
2
the root. Similar sensors have already been used to describe
[O ] distribution within roots (Armstrong et al., 1993, 1994;
2
Ober and Sharp, 1996; Stepniewski et al., 1998). Oxygen is
chemically reduced at the cathode surface. In the conditions
used in this study, the resulting electrical current was usually
between 1 and 200 pA and proportional to [O ] at the tip of
2
the microelectrode. Overall, response time was about 1 s, offset
signal (i.e. at 0% O ) was lower than 15 pA, sensitivity was
2
greater than 5 pA per % of [O ] change and tip diameter was
2
about 50 mm. Electrical current was measured using a picoammeter ( Keithley 487, Cleveland, Ohio, USA). After calibration
of the microelectrode at 0, 20 and 100% [O ], the root system
2
embedded in the agar medium was placed into position
(Fig. 1A). The microelectrode was then inserted perpendicular
to the root surface at the observed boundary between the cap
meristem and the quiescent centre (Fig. 1B). Displacements
were made with a motor-driven micromanipulator (Märzhäuser,
Steindorf-Wetzlar, Germany), which positioned the electrode
tip with an accuracy of 10 mm. The electrode tip and the root
were examined with a microscope throughout the experiment.
Each measurement was taken within a Faraday cage in a
laboratory at 292–293 K. After each [O ] profile, root diameter
2
was measured using a calibrated eyepiece with ×25 or ×50
magnification. For [O ] profiles, 15–20 points per profile were
2
regularly spaced with 10 points located within the nearest
500 mm zone surrounding the root. The last point of the profile
was taken in contact with the root surface.
Preliminary tests evaluated the bias that may result from the
B
Fig. 1. Experimental design for the estimate of local O -consumption:
2
(A) Overview of the whole experimental design with the plant (1) and
its root system suspended in an agar layer (2). A hole (3) was made in
the agar at least 10 mm away from the root in order to observe the
root around the O measurement point. In order to have a sharp image
2
in the binocular field, a histological lamella was applied on the
corresponding vertical agar surface. The tip of an O -microelectrode
2
(4) was monitored with the micro-manipulator (5). Electrical current
intensity, which is proportional to [O ], was recorded with a pico2
ammeter (not shown). (B) Root suspended in the agar gel with O 2
microelectrode tip (20 mm in diameter) at the boundary between the
cap and the quiescent centre, as they can be observed with the
binocular (×25).
radial O diffusion assumption. For these preliminary experi2
ments, [O ] was recorded at every 50 mm during penetration of
2
the microelectrode until the root surface was reached. For plant
no. 1, it was verified that the estimates of microbial constant k
and root respiration R
did not depend on the portion of the
root
oxygen distribution profile around the root used for fitting the
model (the fitting area thickness, Fig. 3). For plant no. 2, R
root
was estimated on most meristems of the root system using the
same microelectrode over a period of 2 d. Preliminary experiments showed that, in these experimental conditions, the Prunus
plants presented no diurnal variations in R
and did not have
root
gas flux from shoot to roots (Bidel et al., 1999). It was thus
possible to compare R
data based on [O ] profiles recorded
root
2
at different times. Spatial progressions in the meristem were
recorded with a calibrated ocular micrometer. Root growth of
plants 2 and 3 was estimated from the deplacement of the apex
between two dates using capillaries planted in the agar to locate
the apex precisely at a particular date.
758 Bidel et al.
Mathematical analysis of O depletion within the root
2
Under steady-state conditions, radial O diffusion within the
2
root can be described by the following equation (Armstrong,
1979):
A
B
1
∂[O ]
∂
2 −MR
0= ×
D
r
(3)
O2−root ∂r
root
r ∂r
where D
is the coefficient of O diffusion within the root
O −root
2
tissue (m22s−1), and MR
is the rate of O consumption by the
root
2
root when [O ] is non-limiting (mol O m−3 tissue s−1).
2
2
Successive concentric layers with their own diffusivity were
proposed for simulating oxygen distribution within roots
composed of differentiated tissues (Armstrong and Beckett,
1985, 1987). As differentiation has not already occurred at the
apex (i.e. in the meristem and the subsequent elongating zone),
root tissues were assumed to behave as a radially homogeneous
porous medium. It was characterized by an average oxygen
diffusivity that takes into account both transfer in the
liquid phase and transfer in the gaseous phase within intercellular spaces filled with gas. As no aerenchyma was observed
in the cortex, connectivity of air spaces for longitudinal
transfer were supposed not to be of significance. It was
assumed that D
does not vary with root thickness and
O2−root
MR
remains constant
as long as O is locally available ( Van
root
2
Noordwijk and de Willigen 1984; De Willigen and van
Noordwijk, 1989).
In order to compare R
between meristems of different
root
sizes, a normalization procedure was used, which enabled us to
compare R
when O concentrations at the root surface (i.e.
root
2
[O ] , mol m−3) are different. Equation (3) can thus be
2s
transformed:
0=
with
A
B
∂dO d
MR
1 ∂
2 −
root
drd
drd ∂drd
∂drd
D
O2−root
(4)
If the MR /D
ratio is identical in different locations,
root O2−root
checking for a unique
relationship between the local root
respiration measured at various points in a root system and
the normalized local root radii dr d may be a means of
root
investigating whether R
variability results from limited O
root
2
diffusion or not. This is illustrated in Fig. 2, where the
relationship between R
and dr d radius for contrasted
root
root
MR
and D
values was plotted, corresponding to
root
O2−root
young (e.g. meristem and expanding zones) and mature segments
zones (e.g. primary and woody zones) having (i) high respiration
and low O diffusion coefficient and (ii) low respiration and
2
high O diffusion coefficient, respectively. R
at the meristem
2
root
level was analysed as a function of the nomalized radius drd
for plants 2 and 3. In this analysis, the results of Bidel (Bidel,
1999) on R
in root segments along the taproot of another
root
45-d-old Prunus (plant 4) were included.
Meristem dimensions and volumetric growth rate
Apical diameter was measured at the boundary between cap
and quiescent centre. The length h
and the apical diameter
mer
d
were measured with a graduated eyepiece micrometer
mer
(magnification ×50). The meristem volume V was estimated
mer
according to Barlow and Rathfelder, assuming its shape to be
one half of a spheroid (Barlow and Rathfelder, 1984):
V
A B
2
d
2
= p×h × mer
mer 3
mer
2
(8)
The volume of tissue produced between two measurement
times about 5 h apart (DV ) was assumed to be equal to the
difference between the meristem volumes at these two times.
However, the volume due to root elongation l (m) was also
added. This last term was estimated assuming conical root
r
[O ]
dO d= 2 and drd=
2
[O ]
√[O ]
2s
2s
This normalization procedure requires defining a relationship
between dO d and drd solely dependent on the normalized root
2
radius dr d as long as the MR /D
ratio is constant.
root
root O2−root
R
can then be expressed as the product
of the fraction of
root in aerobic conditions by maximum respiration rate
tissue
(MR ). It may be estimated by using either actual or
root
normalized radii:
R
=MR ×
root
root
A
B
A
dr
d3−dr d3
r 3−r 3
root/
0 =MR × root
0
root
dr d3
r 3
root
root
B
(5)
where r is the radius under which anoxic conditions prevail (m).
0
There is no anaerobiosis within the root (i.e. dr d=0) as
0
long as the normalized root radius dr d is lower than a critical
root
normalized radius dr d. When it becomes higher, however,
c
anaerobiosis may appear:
S
4D
O −root
(6)
MR2
root
For roots where normalized radii drd>dr d, r is the solution
c
0
of the following equation (Glinski and Stepniewski, 1990):
dr d=
c
0=
C
D
(dr d2−dr d2)
2dr d2
0
root −
0 (Ln(dr d )−Ln(dr d )) +1
0
root
dr d2
dr d2
c
c
where r can be estimated by an iterative fitting procedure.
0
(7)
Fig. 2. Specific respiration rate of the root (R ) as a function of the
root
root normalized radius dr d. For given D
and MR
values,
root
O2−root
root
the specific O -consumption of the meristem
is equal to MR
2
root
regardless of the tissue, as long as the normalized root radius is small
enough. As the normalized root radius becomes higher than a critical
value dr d, anaerobic conditions prevail in a central cylinder of
c
normalized root radius dr d. The external part of the root remains well
0
aerated, i.e. R
is equal to MR
in the external zone. R
of the
root
root
root
whole root section is then estimated by equation (5). Curve A is for an
active young root with D
=1.0×10−11 m2 s−1 and MR =
O2−root
root
5.0×10−2 mol m−3 s−1, Curve
C is for a root segment with a large
intercellular porosity (D
=2.0×10−8 m2 s−1) and low respiration
O2−root
(MR =2.5×10−3 mol m−3
s−1), and Curve B is for an intermediate
root
situation with D
=1.0×10−9 m2 s−1 and MR =1.0×10−2
O2−root
root
mol m−3 s−1.
Respiration of root meristems 759
segments of radii r and r at their two ends:
1
2
1
DV={DV }+ p×l× r2+r r +r2
mer
2 12 1
3
Meristem histological treatments
The apical 10 mm long root segments were excised, washed in
a phosphate buffer and immediately fixed in glutaraldehyde.
They were then dehydrated and embedded in metachrylate resin
Technovit 500 (Heraeus Kulzer GmbH, Philipp-Reis-S trasse
8/13 D-61273 Wehrheim /Ts) as described previously (Bidel
et al., 1999). Microtome sections (0.5–3.0 mm) were stained
using the periodic acid–Schiff ’s (PAS) reaction followed by
toluidine blue-O-( TOB) ( Varney and McCully, 1991). They
were mounted on slides covered with Histolaque (LaboModerne, 75015, Paris). Photomicrography used Kodak 160 T
film and an Olympus Vanox microscope. The mean length of
the meristematic zone was estimated as the distance between
the cap junction and the nearest cell mitosis in the central
cortex (Barlow et al., 1991; Barlow, 1992).
Similarly, the early first-order lateral meristem (M17) was
33 times larger than the second-order lateral meristem
(M13), but it only consumed O 11 times faster. Second2
order lateral meristems were larger and consumed more
O (M10, M7) than some first-order laterals ones. The
2
largest second-order lateral meristem (M15) exhibited an
R
similar to first-order lateral meristems of comparable
root
size. Some first- and second-order lateral meristems had
very low R
(M24, M25, M26). Unfortunately, [O ]
root
2
profile measurements were not taken on the eight first
order lateral meristems. Nevertheless, morphometric
measurements made it possible to estimate that their
respiration rate would hardly reach 15×10−14 mol
O s−1. This estimate was calculated by multiplying the
2
measured meristem volumes by the highest R
estimate
root
for first-order lateral meristems. Consequently, at 15 d,
the taproot meristem respired more O than the sum of
2
all the other meristems in the whole root system.
Results
Correlation between respiration rate and volumic root
growth
G
A
BH
(9)
Reliability of meristem respiration estimates
Even for meristems with low R , the decrease in [O ]
root
2
between the agar surface and the root surface corresponded to an equivalent decrease in the O fraction in
2
air greater than 4%: [O ] at the meristem surface corre2
sponded to O fractions in air between 17.6% and 3.8%.
2
The lower values corresponded generally to high microbial
respiration around the root (Table 1). The electrode signal
drift was never greater than 2%. Simulating numerical
experiments by adding noise to a perfect signal showed
that a random noise of 3% on the microelectrode signal
yields an estimate of R
to the nearest 10% (data
root
not shown).
At first sight, the estimated R
and the constant k
root
measuring microbial respiration did not depend on the
fitting-area thickness, i.e. the maximum cylinder volume
of gel used for model fitting (Fig. 3). Microbial activities
in the agar gel markedly varied between meristems,
with k ranging between 2.1×10−11 and 2.3×10−7 mol
O m−2 s−1.
2
Map of meristem respiration in the 15-d-old plant
Meristems of plant no. 2 grown in agar differed both in
size and respiration rate ( Table 1; Fig. 4). The volume of
the taproot meristem was about 0.680 mm3, while meristems of first-order laterals ranged between 0.012 and
0.143 mm3. The volumes of the second-order lateral meristems and the meristems from the distal short-time growing
laterals occupied between 0.0002 and 0.013 mm3. The
taproot meristem (M1) was 3644 times larger than the
smallest second-order lateral meristem (M23), but it only
consumed O 340 times more rapidly. R
was thus
2
root
approximately ten times lower for larger meristems.
Meristem progression in agar is the result of both the
process of cell elongation in the expanding zone and cell
division in the meristem. The growth rate was roughly
correlated with the meristem R
( Fig. 5). These data
root
made it possible to estimate a maintenance respiration of
5×10−14 mol O s−1 (i.e. the limit of R
when the
2
root
meristem progression tended to zero). This value is less
than 1/8 of the maximum respiration rate at the meristem
level. According to the linear regression calculated
between the volumetric elongation rate and R
shown
root
in Fig. 5, O efficiency was 8.62×10−4 mol O g−1 dry
2
2
matter. This allowed calculation of the construction cost,
1/Yg (ratio of the total weight of substrate consumed to
the weight of tissue produced ). In agreement with the
estimate of 1.02 for the whole tomato root system, (Gary
et al., 1998) 1/Yg was calculated here to be 1.045 g
equivalent glucose g−1 dry matter.
Relationship between the respiration rates and the meristem
radii: normalization procedure
Since R
appeared related to the size of the meristem,
root
respiration estimates made on plants 2 and 3 were plotted
as a function of the meristem radius at the measurement
level (Fig. 6A). Results in R
measured on segments
root
and the taproot meristem of a 45-d-old Prunus persica,
(Bidel, 1999), were also included in this figure and referred
to as plant 4. The general trend is a hyperbolic curve. To
account for [O ] at the root surface, which may differ
2
between meristems, the normalization procedure, previously described in the Materials and methods section,
was applied to this data set ( Fig. 6B). By using the
normalization procedure, (i) a distinction was made
between segments and meristems, because of the lower
760 Bidel et al.
Table 1. Root and microbial respiration at the root tip of Prunus persica (L.) Batsch plant no. 2
.
Number
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
M22
M23
M24
M25
M26
Order
1
2
2
2
2
3
2
2
3
2
2
2
2
3
2
2
2
2
3
2
3
2
3
2
2
3
Meristem geometry
Meristem respiration
Radius
(mm)
Length
(mm)
Volume
(10−3 mm3)
Specific
(10−3 mol m−3 s−1)
Linear
(10−10 mol m−1 s−1)
Bulk
(10−14 mol s−1)
380
137
137
137
141
78
68
117
93
88
68
98
98
59
88
68
98
107
44
78
78
83
39
107
78
39
2243
332
332
312
341
176
254
176
273
127
176
254
215
98
254
137
332
234
98
449
234
449
59
215
198
312
679.1
12.9
12.9
12.2
14.3
2.2
4.6
5.0
4.9
2.0
2.8
5
4.3
0.7
4.1
1.3
6.6
5.6
0.9
5.7
2.9
6.4
0.2
5.2
2.5
1.0
3.0
12.3
12.1
10.0
7.8
24.0
15.7
9.0
13.5
14.2
13.8
0.3
11.1
30.3
12.4
19.1
9.3
7.6
36.9
11.6
11.5
8.2
32.5
4.2
4.9
8.6
13.8
7.2
7.1
5.9
4.9
4.6
4.2
3.9
3.6
3.4
3.3
10.4
3.3
3.3
3.0
2.8
2.8
2.7
2.2
2.2
2.2
1.8
1.6
1.5
0.9
0.4
205.9
15.9
15.7
12.2
11.1
5.4
7.1
4.5
6.6
2.9
3.9
42.9
4.7
2.1
5.1
2.5
6.1
4.3
1.4
6.6
3.4
5.3
0.6
2.2
1.2
0.9
Microbial
constant k
10−10 mol m−2 s−1)
Root surf.
[O ]
2s
(mol m−3)
0.2
2216
1.4
2.1
1159
20.6
1283
0.0
20.6
940
1628
5.2
549
2006
256
156
1245
877
36
0.6
828
1230
676
643
166
1645
0.184
0.147
0.139
0.203
0.121
0.108
0.070
0.205
0.147
0.096
0.139
0.196
0.195
0.239
0.112
0.129
0.185
0.202
0.143
0.165
0.145
0.056
0.228
0.243
0.261
0.115
Rate of O2 consumption (10_14 mol O2 .s_1)
Respiration of root meristems 761
Meristem growth rate (mm3 .day_1)
Fitting area thickness (mm)
Fig. 3. Estimates of the specific root respiration R
and the constant
root
k describing microbial respiration within the gel, by fitting a model of
O diffusion and consumption to an experimental [O ] profile recorded
2
2
on the vertical of the taproot meristem of Prunus persica (L.) Batsch
no. 1 (15-d-old plant and 10 fully expanded leaves, growth in agar gel ).
Fig. 5. Relationship between meristem growth, expressed as the volumic
rate of tissue production (mm3 d−1), and O consumption of the
2
meristem (mol O s−1) (r2=0.90). The efficiency in root respiration was
2
close to 6.62×10−4 mol O g−1 dry matter.
2
Fig. 4. Root system architecture of Prunus persica (L.) Batsch no. 2 (15-d-old plant and 12 fully expanded leaves, growth in agar layer). Meristem
identification is numbered according to decreasing R
values. (The same identification was used in Table 1. Meristems 1, 2, 3, 20, and B were
root
in Fig. 7).
[O ] at the segment root surface (Bidel, 1999) and,
2
possibly, (ii) the residual variability of R
was reduced
root
close to the overall trend (i.e. the hypothetical hyperbolic curve).
Considering the limited number of measurements and
the contrast between apex and segments for both the
porosity and R , the normalized model, equations (5),
root
(6) and (7), was fitted separately to meristems and
segments data thus yielding two experimental relationships between R
and normalized root radius (Fig. 6B).
root
The model fitted the data well and no positive or negative
biases were observed. D
and MR
were estimated
O2−root
root
to be equal to 9.2×10−12
m2 s−1 and 5.7×10−2 mol
O m−3 of tissue s−1 for meristems, and 2.36×
2
10−7 m2 s−1 and 2.53×10−3 mol O m−3 of tissue
2
s−1 for segments. D
was expressed for O diffusing
O2−root
2
in a liquid phase: [O ] at the root surface equalled actual
2s
O concentration within the agar gel, and [O ] within the
2
2
root was expressed assuming that O diffuses in water. If
2
O diffusion happened in the air-filled intercellular spaces,
2
the corresponding O diffusion coefficient could be estim2
ated by multiplying the previous values by the coefficient
of O solubility in water (approximately 0.034 at 293 K ),
2
because the previous [O ] gradient would be divided by
2
this value to obtain [O ] gradient in a gas phase.
2
Therefore, the diffusion coefficients would then be
762 Bidel et al.
A
Discussion
Respiration estimates
B
Fig. 6. Specific respiration rate (R ) of meristems of first, second and
root
third orders of Prunus persica (L.) Batsch no. 2, meristems of Prunus
no. 3 at various dates, and segments and meristem of the taproot of
Prunus no. 4 (45-d old, 26 leaves fully expanded, growth on a Nylon
mesh and inclusion of the root system in an agar layer 1 d before
measurements). (A) R
is plotted versus the actual root radius r ;
root
root
(B) R
is plotted versus the normalized root radius dr d. The model
root
root
of O diffusion and consumption was fitted to the whole set of
2
meristems, giving us the estimates: MR =5.7×10−2 mol O m−3 s−1
root
2
and D
=9.2×10−12 m2 s−1 for O diffusion in a liquid phase.
O2−root
2
Fitting the
model to the set of experimental segment respirations gave
us the estimates: MR =2.53×10−3 mol O m−3 s−1 and D
=
root
2
O2−root
8.0×10−9 m2 s−1, considering [O ] gradient in the intercellular air-filled
2
space (i.e. expressed in an air phase).
3.1×10−13 and 8.0×10−9 m2 s−1 for meristems and segments, respectively.
Histology
Variability in the length of the meristematic area is greater
than in the diameter ( Fig. 7A–E). Some meristems lost a
part of their cap embedded in agar gel during sampling
(Fig. 7C, D). Smaller meristems with higher R
were
root
less stained by meristematic indicators, such as PAS-TBO
or methyl green (Gahan, 1984). These presented cap cells
without amyloplasts. Longitudinal histological sections
revealed that it finally reconstituted a new root cap that
was not always typical in size and staining.
The model of radial O diffusion within the agar gel
2
apparently fitted well with measurements, even when [O ]
2
measurements were taken close to the root tip, as long as
the fitting area thickness did not exceed 500 mm (and
sometimes more as in Fig. 3). The resulting estimates of
R
and constant k that characterized the microbial
root
respiration did not depend on the fitting area thickness
in this domain ( Fig. 3). Furthermore, the lack of variation
in the parameter k with changes in the fitting area
thickness would indicate that microbial respiration in the
agar gel can be modelled by a function proportional to
the (1/r) ratio, as already suggested (Højberg and
Sørensen, 1993; Bidel, 1999).
Additional assumptions were made to estimate total
O consumption in each meristem. Their shape was
2
assumed to be spheroid, and all their tissues were assumed
to have a specific respiration rate equal to the estimated
R
value. There was generally a good agreement
root
between the observed length of the translucent zone of
the meristem and the length of the meristematic area on
histological sections. However, there was not good agreement for the two largest meristems (the taproot meristems
of plants no. 2 and no. 3) out of the 27 meristems for
which R
was measured. It was only possible to obtain
root
18 histological sections and these were not always in the
axial plane. For these, volume estimations based on
microscopic observation were made. Therefore, the O
2
consumption of the taproot meristem was probably
underestimated by about 10–15%. Despite these uncertainties, the results of this study are indicative of the
relative activity of the set of meristems in an entire
root system.
R
of the taproot meristems of plants 1, 2 and 3 were
root
equal to 5.3, 13.8, and 7–8.9 nmol O m−1 s−1. These
2
values were in fairly good agreement with measurements
taken on excised root tips placed into stirred nutrient
solutions ( Table 2). For these root tips, however, meristem respiration was not discernible from that of the
elongation zone with the ‘excised root’ experimental procedure. No results were found in the literature about
respiration rate of second and third order meristems.
The conditions of low light and very high humidity
probably caused R
meristem values to be lower than
root
those expected for roots growing in a rhizotron under
standard climatic conditions (Pagès, 1995). Moreover,
for the same aerial climate, plants grown in the agar layer
had achieved only one-half to one-third of the growth
rate of plants cultivated in a rhizotron or on nylon mesh
(L Pagès, unpublished results). As an indirect confirmation of this effect of agar gel, meristems that emerged
from the agar layer thereafter progressed quicker than
meristems remaining in the gel. These two facts suggested
Respiration of root meristems 763
Fig. 7. Histological longitudinal sections of the taproot meristem (A) and four first-order lateral meristems (B–E ) stained periodic acid-Shiff ’s
reaction, followed by toluidine blue O. Taproot meristem reconstituted a new cap not fully characteristic. Meristems M2 and M3 (C–D) lost their
cap when removing the root from the agar gel. No aerenchyma were present in the cortex. (Bars: 75 mm). (A) Meristem M1, R =3.0×10−3
root
mol m−3 s−1, (B) Meristem B, (C ) Meristem M2, R =12.3×10−3 mol m−3 s−1, (D) Meristem M3, R =12.1×10−3 mol m−3 s−1. (E ) Meristem
root
root
M16, R =19.1 10−3 mol m−3 s−1.
root
the occurrence of limiting factors for root growth in the
experimental design, such as O depletion. Other adverse
2
factors such as a progressive dehydration, acidification
and mineral depletion of the agar around older root
segments may also have reduced the meristem growth.
Respiration rate as an indicator of meristem activity
Since meristem respiration rate appears significantly
correlated to root elongation rate (r2=0.90), it may be
considered as a growth indicator. However, not enough
764 Bidel et al.
Table 2. Respiration of excised root tips, measured with Clark O -electrode in nutrient solutions
2
Reference
Temperature
(°K )
Age
(d)
Root tip length
(mm)
Bulk root tip respiration
(nmol O m−1 s−1)
2
Saglio et al. (1983)
Brouquisse et al. (1992)
James (1994)
Williams and Farrar (1992)
298
298
293
301
3
3
3
5
5
3
3
5
6–8
20–25
18–21
6–13
points were plotted to ensure that the relationship was
strictly linear. The residual variability may partially result
from measurement errors on meristem diameter and
length, and biased shape assumption. The taproot meristem of the 15-d-old plant consumed O at a rate more
2
than ten times higher than that of its nearest laterals
( Table 1), suggesting that it could deprive these laterals
of carbohydrates and reduce their activity. The total
active tissue in the root tip, including both the elongation
zone and the meristem, would probably have increased
the magnitude of competition. The length of the elongation zone of roots with small meristems was very small
compared with roots with larger meristems, which is
visible when Fig. 7A–E are compared.
The observation that the taproot meristem has the
greatest O consumption suggests that it is also the
2
strongest carbohydrate sink. This is consistent with previous reports in the literature, although such data are rarely
quantitative ( Webb, 1977; Daie, 1985; Schulz, 1994). For
many species, the more developed the main axis is, the
more it seems to inhibit growth of its laterals (Atzmon
et al., 1994). Furthermore, the removal of the root tips
is known to stimulate the growth of the youngest and
nearest laterals greatly ( Wightman et al., 1980; Atzmon
et al., 1994a, b).
Oxygen diffusion within root tissue as a factor limiting
meristem respiration
The theoretical model based on O diffusion and con2
sumption within root tissue is quantitatively in fairly
good agreement with the experimental results, especially
in view of the experimental difficulties and the theoretical
approximations made for obtaining these values. Two
independent reasons suggested that [O ] diffusion and
2
consumption within the root was really the main factor
involved in R
variations between the meristems. (1)
root
No discrepancy was observed between experimental
and simulated data ( Fig. 6B). A discrepancy would be
expected if carbohydrate supply was selectively limiting
respiration rate in aerobic tissues as a function of meristem size. (2) The estimated O diffusion coefficients within
2
the root tissue were consistent with the real structure seen
in histological sections.
The O diffusion coefficient in pure water is about
2
2.09×10−9 m2 s−1 at 293 K and decreases in saline water.
This value is 4 orders of magnitude lower than the
coefficient in air. Given that the calculated O diffusion
2
coefficient at the meristem level is about 9.2×
10−12 m2 s−1, O must have moved there as a solute in a
2
liquid phase. The root:water O diffusion coefficient ratio
2
was about 0.004, indicating that the cell walls and plasma
membrane greatly reduced O diffusion. It was assumed
2
by other authors that the diffusion coefficient of the
cell walls was about 6.3×10−10 m2 s−1 (Armstrong et al.,
1994); these results suggest an even greater resistance of
the cell walls and membranes. In the meristem, it seems
unrealistic to consider O diffusion in the inter2
cellular pore space, because it is difficult to observe such
a space in histological sections. Conversely, it may be
considered that only O moves in the air-filled intercellular
2
pore space at the segment level, because O diffusion in
2
pure water is lower than the estimated O diffusion
2
coefficient within the root. Considering the O diffusion
2
coefficient in air (2.01×10−5 m2 s−1 from Jaynes and
Rogowski, 1983), the root segment:air O diffusion
2
coefficient ratio would be equal to 0.0004. Using
Buckingham’s model (Buckingham, 1904) to relate this
ratio to the air-filled porosity of porous media, it was
estimated that the air-filled intercellular pore space actually involved in O diffusion corresponded to approxi2
mately 2% of the root volume. This is only a rough
estimate, because the porous media:air O diffusion
2
coefficient ratio greatly depends on the actual geometry
of the intercellular space (Cousin et al., 1999). The
estimate of 2% is in the range of published values from
various methods, such as picnometer measurements:
values of 2, 3 and 4–9% for Festuca, barley, and tomato,
respectively, have been reported (Glinski and Stepniewski,
1990); and values between 1% and 4% for bean have
been reported (De Willigen and Van Noordwijk, 1989).
Assuming a constant D
for all the meristems of
O −root
the root system, the present2 diffusion model suggests the
existence of a unique maximum respiration rate value
MR . This corresponds to a potential for R
in the
root
root
meristem tissues. An unique potential R
has still to be
root
confirmed by different measurements on other plants. In
theory, this would depend on plant species and environmental conditions, such as temperature. For the 15-d-old
plants, a unique potential R
would also indicate that
root
the activities in all meristematic zones in aerobic conditions were not limited by the carbohydrate supply. This
could have resulted from the presence of cotyledons and
the slow development of the plants in agar gel.
Respiration of root meristems 765
These reasons cannot explain, however, the differences
in growth rate between meristems, resulting mainly from
their size. It may be assumed that differences in growth
rates are due to competition for carbohydrates: the supply
of carbohydrate to a meristem would influence its own
diameter. On a short time-scale, both the anatomy and
the O tissue diffusion properties of the meristem define
2
aR
that could be satisfied if carbohydrate availability
root
made it possible. When the meristem is supplied by lower
carbohydrate availability caused by competition with
other organs, a lower respiration rate and a lower mitotic
activity could be expected. Consequently, a reduced
number of cell ranks could be generated as reported for
meristems in vitro ( Feldman and Torrey, 1975; Barlow
and Adams, 1989). That may explain daily variation in
size of the apical diameter in parallel to photosynthesis
activity in vivo for Hevea brasiliensis Müell. Arg. seedlings
plant in the phytotron at constant temperature ( Thaler
and Pagès, 1996a). This would lead to a kinetic adjustment
of the meristem dimensions that minimizes carbohydrate
limitation for aerobic metabolism in a larger time-scale.
The taproot meristem usually increases its size during
the establishment stage on nylon mesh culture with similar
[O ] and temperature. The proposed cylindrical model
2
of respiration simulates an increase of total carbohydrate
consumption by aerobic metabolism with the increase of
the meristem diameter, although the meristem anoxic
fraction increases. The size of excised meristems cultivated
in vitro was positively correlated with hexose supply,
which also governed the complexity of the vascular
pattern of the formed axes ( Feldman and Torrey, 1975;
Scadeng and MacLeod, 1976).
When temperature increases, thinner lateral roots grow
faster than the main roots (MacDuff et al., 1986; Gregory,
1986). Growth rate in soybean (Glycine max [L.] Merr.)
taproots decreases as temperature increases, whereas lateral growth rate remains steady (McMichael and
Quisenberry, 1993). Based on the results presented here,
it is suggested that these different responses of root types
to temperature may result from the effect of O depletion
2
on meristematic activity. A 10 K increase in temperature
slightly affects O diffusion (Renault and Stengel, 1994),
2
whereas root respiration increases exponentially with a
Q between 1.5 and 3.0 (Lambers et al., 1996; Sprugel
10
et al., 1994; Amthor, 1989). Consequently, hypoxia within
the root may appear and/or increase as temperature
increases (Glinski and Stepniewski, 1985). The thinner
lateral roots would be expected to be less affected by
hypoxia than the wider taproot as temperature increased,
and therefore have a greater temperature optimum for
elongation.
Conclusions
As far as is known, this is the first work that describes a
map of meristem O consumption for a root system
2
architecture. The experimental procedure and the related
device appeared to be adequate for investigating root
activity with low disturbing effect for seedling, although
it may affect meristem respiration because of [O ] limita2
tion at the root surface. The proposed normalization of
the data (i.e. the definition of a normalized root radius
equal to the actual radius:square root of the surface [O ]
2
ratio) made it possible to discuss the data, regardless of
the actual effect of the agar gel on root respiration.
Diffusion of O within the root tissues appeared to be
2
the main limiting factor for meristem respiration. The
proposed model of O radial diffusion and consumption
2
within the root tissues was in fairly good quantitative
agreement with the experimental data. It enabled an
estimatation of a unique O diffusion coefficient and a
2
unique maximum specific respiration rate for all the apical
meristems of the root systems studied. Due to the
uniqueness of these parameters, using this theoretical
approach would also make possible the definition of the
minimum [O ] at the root surface to avoid O limitation,
2
2
and the estimation of the meristem respiration rates for
roots growing in aerated conditions (i.e. 20% for the O
2
fraction). The uniqueness of these parameters would also
suggest that the activity of the central volume of root
tissue in anaerobic condition is not limited by the
carbohydrate supply. This might be caused by kinetic
adjustment of the meristem dimensions to the local carbohydrate supply.
Acknowledgements
This work was carried out in the Soil Science Unit at INRA,
Avignon (France). We thank NP Revsbech ( University of
Aarhus, Denmark) and the members of his Laboratory for
training one of us in the construction of O microelectrodes.
2
We also thank M Dever of the Language Service, INRA, for
reviewing the English version of the manuscript, S Parry for
helping to construct O microelectrodes and V Serra for young
2
peach trees cultivation. The authors wish to thank JL Poessel
and V Restier (INRA, Avignon) for their advice and help with
logistic assistance in preparing the micro-sections, and M
Chevalier (INRA, Angers) for microscopy.
766 Bidel et al.
Appendix
List of symbols
Symbol
Definition
Unit
[O ]
2
[O ]
2s
dO d
2
r
r
root
r
0
drd
dr d
root
dr d
0
dr d
c
D
O −root
D 2
O2−gel
R
root
LR
root
MR
root
k
Actual O concentration
2
O concentration at the root surface
2
Normalized O concentration
2
Radial position
Actual root radius
Radius under which anoxia prevails
Normalized radial position
Normalized root radius
Normalized radius under which anoxia prevails
Critical normalized radius for anoxia
O diffusion coefficient within the root
2
O diffusion coefficient within the agar gel
2
Specific root respiration
Root respiration expressed by root length
Volumic root respiration when [O ] is not limiting
2
Constant used to describe an hyperbolic decrease of microbial respiration
k/r
mol m−3
mol m−3
no unit
m
m
m
m mol−0.5
m mol−0.5
m mol−0.5
m mol−0.5
m2 s−1
m2 s−1
mol O m−3
2
mol O m−1
2
mol O m−3
2
mol O m−2
2
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