Plant Physiol. (1992) 100, 1891 -1900
0032-0889/92/1 00/1891/1 0/$01 .00/0
Received for publication April 6, 1992
Accepted August 7, 1992
Effects of Glucose Starvation on Mitochondrial
Subpopulations in the Meristematic and Submeristematic
Regions of Maize Root
Ivan Couee*, Murielle Jan, Jean-Pierre Carde, Renaud Brouquisse, Philippe Raymond, and Alain Pradet
Institut National de la Recherche Agronomique, Centre de Recherche de Bordeaux, Station de Physiologie
Vegetale, BP81, 33883 Villenave d'Ornon Cedex, France (I.C., M.J., R.B., P.R., A.P.); and Universite Bordeaux 1,
Laboratoire de Physiologie Cellulaire Vegetale, Unite Associee Centre National de la Recherche Scientifique 568,
33405 Talence Cedex, France (I.-P.C.)
ABSTRACT
Mitochondria isolated from 3-mm long maize (Zea mays L. var
Dea) root tips were found to be heterogeneous on Percoll density
gradients. The ultrastructure of these isolated mitochondria correlated well with that of mitochondria observed in situ and was
consistent with the existence of mitochondria at different stages of
maturation during cell development. The mitochondria of higher
density presented an ultrastructure with many cristae and a dense
matrix. These mitochondria showed classic respiratory properties,
although with low ADP/O ratios. In contrast, the mitochondria of
lower density showed few cristae and a clear matrix and did not
seem to be fully functional because their rate of respiration was
low and showed weak respiratory control. Lower- and higherdensity mitochondria were shown to be differentially affected
during the first stages of glucose starvation. The higher-density
mitochondria from glucose-starved maize root tips retained the
ultrastructure and most of the respiratory properties of nonstarved
mitochondria, whereas lower- and intermediate-density mitochondria were-absent in the mitochondrial preparations from glucosestarved maize root tips and were not observed in situ. Quantitatively, there was a decrease of the total mitochondrial pool when
expressed as the amount of mitochondrial protein per root tip.
However, this decrease affected low- and intermediate-density
mitochondria, but not higher-density mitochondria. Thus, it was
shown that a significant pool of functional mitochondria is maintained in maize root tips during the first stages of glucose starvation. The reasons for these apparently selective effects of glucose
starvation on mitochondria are discussed in relation to effects on
mitotic and differentiation processes.
breakdown of lipids and proteins (2, 4, 7), and the more or
less marked disappearance of certain organelles (2, 4, 15, 21).
Joumet et al. (15) gave evidence that, in sycamore cells,
the progressive decrease in the uncoupled rate of 02 consumption could be ascribed to the decrease of the mitochondrial pool, as determined by the levels of intracellular cardiolipin and Cyt aa3. In excised maize (Zea mays L.) root tips,
the decrease of enzyme activities specific to the mitochondrial
matrix and the mitochondrial inner membrane also suggested
that carbohydrate starvation induced the degradation of mitochondria (4). However, in this latter case, there was not a
strict correlation between the decrease in uncoupled respiration and the decrease in mitochondrial enzyme markers (4).
Thus, the situation appears to be more complex in the root
tip, which may reflect the complex cellular organization of
this structure consisting of meristematic cells and of different
types of more differentiated cells (6). Cell development is
known to be associated with a parallel maturation of mitochondria (8). Thus, in maize root tips, earlier studies (5) have
shown the presence of mitochondria at different stages of
maturation. In the present work, preparations of Percollpurified mitochondria with distinct ultrastructural and metabolic properties were isolated from maize root tips and used
to assess the qualitative and quantitative effects of glucose
starvation on the different types of mitochondria. These
results obtained in vitro were compared with EM observations in situ.
Carbohydrates are the main respiratory substrates in plant
cells (1). Under certain circumstances such as lower light
intensity, lower temperature, or biotic stress, which lead to a
significant decrease in photosynthesis, the supply of carbohydrates to sink tissues may become limiting (12, 16; see also
4). The effects of carbohydrate limitations have been studied
in plant systems subjected to long-term carbohydrate starvation (4, 14, 15, 17, 18). Such long-term starvation has been
shown to trigger a coordinated sequence of events, with the
depletion of intracellular carbohydrate content and a subsequent decrease in respiration (2, 4, 15, 17, 18, 20), the
MATERIALS AND METHODS
Plant Material and Glucose Starvation Treatment
Germination of maize seeds (Zea mays L. var Dea) was
carried out at 250C in the dark for 3 d between sheets of
filter paper soaked in a mineral nutrient medium, as described
by Saglio and Pradet (18). The 3-mm-long tips of the seminal
roots were then excised and either immediately used for
analysis or incubated for glucose starvation treatment. In this
case, the excised root tips were incubated at 250C in the
mineral nutrient medium supplemented with 1% (v/v) of the
antibiotic and antimycotic mixture A 7292 from Sigma Chemical Co. and 0.1 M Mes (pH 6.0). A gas mixture containing
50% 02 and 50% N2 was continuously bubbled through the
incubation medium to maintain a partial oxygen pressure
1891
1 892
COUEE ET AL.
above 35 kPa, which is the critical oxygen pressure for maize
roots in aqueous solutions (19). When control experiments
were carried out in the presence of 0.2 M glucose, a significant
elongation of the excised root tips occurred and, thus, 3-mm
long tips were reexcised.
Purification of Mitochondria
Mitochondria were prepared by a modification of the procedure described by Douce et al. (9) from 3-mm long tips
freshly excised from the seminal roots of 3-d-old maize
seedlings or from 3-mm long tips that had been excised from
the seminal roots of 3-d-old seedlings and then subjected to
48 h of glucose starvation treatment. All homogenization and
isolation procedures were performed at 0 to 40C. Excised root
tips (1000) were ground with a mortar and pestle in a medium
containing 25 mm Mops-KOH, pH 7.8, 1 mm sodium EDTA,
5 mM KCl, 300 mm mannitol, 8 mM cysteine, 0.1% (w/v) fatty
acid-free BSA, and 0.6% (w/v) soluble PVP. The homogenate
was filtered through two layers of muslin and two layers of
Miracloth (Calbiochem) and the resulting crude extract was
centrifuged at 1,000g for 10 min. The supematant was centrifuged at 10,000g for 15 min. The resulting pellet was
resuspended in a washing medium containing 10 mm
KH2PO4-KOH, pH 7.2, 1 mm sodium EDTA, 300 mm mannitol, and 0.1% (w/v) fatty acid-free BSA. This suspension
was centrifuged at 300g for 10 min and the resulting supernatant was centrifuged at 10,000g for 15 min, thus yielding
a pellet of crude mitochondria. This pellet was resuspended
in 1 mL of washing medium and samples of 0.5 mL were
fractionated by isopyknic centrifugation in 13 mL of 26%
(v/v) Percoll in washing medium at 40,000g for 30 min. The
content of the gradient was fractionated by carefully pipetting
out 1-mL fractions. Each fraction was diluted at least 11-fold
with washing medium and centrifuged at 12,000g for 15 min.
The pellets were resuspended in 1 mL of washing medium
and recentrifuged at 10,000g for 15 min. The final pellet was
resuspended in 0.2 mL of washing medium. The density of
Percoll-purified material was determined by comparison with
the migration of density marker beads (Pharmacia).
Analysis of Proteins
Protein contents were determined by the method of Bradford (3) using bovine y-globulin as the standard. The content
of BSA in the extraction and washing mediums, expressed as
the equivalent in bovine -y-globulin, was subtracted from the
total protein content. The added BSA represented 10 to 50%
(w/w) of the total protein measured per sample.
Measurement of Mitochondrial Respiration
Oxygen uptake by mitochondria was measured at 250C
using a Clark electrode system from Hansatech Ltd. Mitochondria (0.2-0.4 mg of protein) were added in 1 mL of a
medium containing 5 ml KH2PO4-KOH (pH 7.2), 10 mm
KCl, 5 mM MgCl2, 300 mm mannitol, and 0.1% (w/v) fatty
acid-free BSA. The 02 concentration in the air-saturated
medium was taken to be 240 ,LM (1 1). Respiratory control and
ADP/O values were calculated according to Estabrook (11).
Plant Physiol. Vol. 100, 1992
Respiratory substrates were made up as concentrated stock
solutions and adjusted to pH 7.2 to maintain the volume and
the pH of the reaction medium. The respiration of succinate
(5 mM) was carried out in the presence of 0.3 mm ATP. The
respiration of NADH was carried out at a concentration of 1
mm. The respiration of 2-oxoglutarate (5 mm) was carried out
in the presence of 0.3 mm thiamine pyrophosphate and 3 mm
malonate. The respiration of pyruvate (10 mM) was carried
out in the presence of 1 mm NAD+, 0.3 mm thiamine pyrophosphate, 0.5 mm CoA, and 0.5 mm malate. The respiration
of citrate (10 mM) was carried out in the presence of 1 mm
NAD+, 0.3 mM thiamine pyrophosphate, and 0.5 mm CoA.
State 3 respiration was triggered by the addition of 0.1 mm
ADP. In each assay, at least two state 3 to state 4 transitions
were performed. The integrity of the outer membrane was
assessed from the accessibility of the inner membrane to
exogenous Cyt c, as described by Douce et al. (10). In this
test, inhibition of Cyt c oxidase was obtained with 200 ZlM
NaN3.
Enzyme Activities
All enzyme activities were assayed spectrophotometrically
at 250C according to previously published methods. All
assays were first performed on blanks. Activities were linear
with respect to time for at least 1 min and were proportional
to the amounts of sample protein added to the assay. The
methods for measuring the activities of fumarase (EC 4.2.1.2),
NAD-IDH' (EC 1.1.1.41), NAD-GDH (EC 1.4.1.2), SDH (EC
1.3.5.1), and G6PDH-6PGDH (EC 1.1.1.49 and EC 1.1.1.44,
respectively) were essentially the same as those used by
Brouquisse et al. (4). The assay of G6PDH-6PGDH and NADIDH in the absence or in the presence of 0.025% (w/v) Triton
X-100 allowed the determination of the immediate activity
and of the latent, membrane-enclosed activity as described
by Douce et al. (9).
EM
The pellets of mitochondria were resuspended in the washing buffer without BSA and fixed with 1.5% (v/v) glutaraldehyde for 1.5 h at 40C. After thorough washing with 100
mM phosphate buffer, pH 7.2, containing 0.3 M mannitol, the
mitochondria were pelleted (10,000g, 15 min) and postfixed
with 1% (w/v) osmium tetroxide in the same medium for 2
h at 40C. These pellets were then treated with 1 % (w/v)
tannic acid (BDH) in phosphate buffer, pH 7.2, for 1 h at
200C. The pellets were covered with 3% (w/v) agar, dehydrated with ethanol and propylene oxide, and embedded in
epon. The root tips were processed according to the same
experimental procedure. Ultrathin sections were collected on
uncoated 600-mesh HT grids (Gilder, Grantham, UK), stained
with uranyl and lead, and then observed with a Philips CM10
electron microscope.
'Abbreviations: NAD-IDH, NAD-specific isocitrate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; HB, heavy band
of material on the 26% Percoll density gradient; LB, light band of
material on the 26% Percoll density gradient; NAD-GDH, NADspecific glutamate dehydrogenase; 6PGDH, 6-phosphogluconate
dehydrogenase; SDH, succinate dehydrogenase.
EFFECTS OF GLUCOSE STARVATION ON ROOT TIP MITOCHONDRIA
RESULTS
Density-Related Structural and Functional Heterogeneity
of the Mitochondria from Nonstarved Maize Root Tips
Crude mitochondria were isolated from nonstarved maize
root tips as described in 'Materials and Methods.' Figure 1A
shows that these mitochondria, as determined from the activity of fumarase, were heterogeneous with respect to their
density on a 26% Percoll density gradient. HBs and LBs
corresponding respectively to fractions 2-3 and fractions 1314 were collected and used for further studies. These HB and
LB preparations had respective densities of approximately
1.056 and 1.042. Marker enzymes of subcellular localization
(Table IA) and respiration studies (Table IIA) showed that
these fractions were enriched in mitochondria.
HB and LB mitochondria obtained from nonstarved root
A.
I
._
0.4
1.6
0.3
1.2 °
0.2
0.8
0.1
0.4
E
I
E
-aZ..
0
:LL
a
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Eo
,-
0.0
0.0
0
3
6
9
12
15
1893
tips were examined by EM (Fig. 2). The pellet of LB mitochondria was highly enriched in intact mitochondria and
contained only a few membrane vesicles, some of which were
of mitochondrial origin. These mitochondria contained a
diluted matrix and few cristae. Some of these mitochondria
were not spherical, but irregularly shaped and encased within
each other. In contrast, HB mitochondria contained a dense
matrix and several elongated cristae, and sometimes a lensshaped dark crystal. The HB fraction also contained some
broken mitochondria and membrane vesicles. In both cases,
the mitochondria showed a mean diameter of 0.7 to 1 ,um.
The mitochondrial ultrastructure was followed in situ from
the meristematic region to the more differentiated tissues
close to the excision area (Fig. 3). In the meristematic cells of
the tip, mitochondria appeared as nearly electron-translucent
areas surrounded by a ribosome-rich cytosol and enclosed in
a double membrane system (Fig. 3a). In the course of cell
differentiation, the mitochondrial matrix became more dense
while a complex system of mitochondrial cristae developed.
Mitochondrial maturation resulted in the presence of typically
differentiated mitochondria near the excision area (Fig. 3b).
Isolated lower-density mitochondria showed poor outer
membrane integrity (Table IA and Fig. 2) and low respiration
rates with weak respiratory control (Table IIA). However,
they showed significant specific activities of mitochondrial
enzymes (Table IIIA). In contrast, isolated higher-density
mitochondria showed a high integrity of the outer membrane
(Table IA). They were able to utilize respiratory substrates
with rates at least 2.2-fold higher and showed specific activities for fumarase and NAD-IDH at least twice higher than
those of the lower-density mitochondria (Tables IIA and
IIIA).
Fraction number
B.
0.4
1.6
0.3
1.2
0
0.2
0.8 5'
E'
0.1
0.4
._S
E
EI
o
._2IL
0.0
0.0
0
3
6
9
12
15
Fraction number
Figure 1. Distribution of fumarase activity and protein on Percoll
density gradients after centrifugation of crude mitochondria isolated
from nonstarved maize root tips (A) and from 48-h glucose-starved
maize root tips (B). Crude mitochondria were isolated from 1000
maize root tips and centrifuged on a 26% Percoll density gradient.
Fractions of 1 mL were collected and assayed for fumarase activity
and protein content after elimination of Percoll. Fraction 0 corresponds to the bottom of the centrifuge tube. HBs and LBs corresponding respectively to fractions 2-3 (A) or 1-2 (B), and fractions
13-14 (A) or 14-15 (B) were collected and used for further studies.
The results show one typical experiment from three concordant
experiments.
Absence of Lower-Density Mitochondria in
Glucose-Starved Maize Root Tips
Crude mitochondria were isolated from 48-h glucosestarved maize root tips and centrifuged on Percoll density
gradients under the same conditions as those used for the
study of nonstarved mitochondria. The main feature of the
density profile (Fig. 1B) was the disappearance of fumarase
activity in the upper regions of the 26% Percoll density
gradient. This phenomenon was related to glucose starvation.
A similar experiment carried out with mitochondria obtained
from excised maize root tips that had been incubated for 48
h in the presence of 0.2 M glucose gave a distribution pattern
sinilar to that of mitochondria from nonstarved roots (data
not shown). Previous studies on the effects of glucose starvation on excised maize root tips have also shown that the
observed effects could be ascribed to glucose starvation rather
than to excision wounding (4).
Marker enzymes of subcellular localization did not show
any clear enrichment in mitochondria in LB material, which
corresponded to fractions 14-15 of the Percoll gradient (Table
IB). Specific activities of mitochondrial enzymes were depleted (Table IIIB) and respiration activity was very low
(Table IIB). EM studies showed that the LB preparation
obtained from glucose-starved root tips did not contain many
intact mitochondria but did contain various membrane vesicles, some of which were of mitochondrial origin with inner
COUEE ET AL.
1 894
Plant Physiol. Vol.
100,
1992
Table I. Homogeneity and Integrity of Mitochondria Isolated from Maize Root Tips prior to Glucose
Starvation (A) and after 48 h of Glucose Starvation (B)
The results are the mean (±SE) of at least three experiments, except in the case of latency values,
where only two results are given. The enrichment was calculated as the ratio of the specific activity
in the preparation to that in the crude extract. Other details are as described in "Materials and
Methods."
HB Preparation
LB Preparation
Enrichment in immediate G6PDH-6PGDH activity
Enrichment in latent G6PDH-6PGDH activity
Enrichment in NAD-IDH activity
Enrichment in fumarase activity
Latency of NAD-IDH activity (%)
Integrity of the outer membrane (%)
0.06 ± 0.01
0.004 ± 0.001
10 ± 3
11 ± 4
80-99
85 ± 4
0.06 ± 0.01
0.0011 ± 0.0001
5±2
4± 1
87-88
45 ± 2
Enrichment in immediate G6PDH-6PGDH activity
Enrichment in latent G6PDH-6PGDH activity
Enrichment in NAD-IDH activity
Enrichment in fumarase activity
Latency of NAD-IDH activity (%)
Integrity of the outer membrane (%)
0.010 ± 0.001
1.2 ± 0.2
6± 1
4± 1
99-99
87 ± 2
0.020 ± 0.002
0.44 ± 0.01
0.20 ± 0.02
0.5 ± 0.4
85-87
0
A.
B.
cristae distinctly attached (Fig. 4). The content of most of
these vesicles was electron transparent. The significant rate
obtained with succinate as respiratory substrate (Table IIB)
confirmed that this vesicular material was at least partially
derived from mitochondria. Oxidation of exogenous Cyt c
was also carried out by this LB material, although with no
barrier to the accessibility of Cyt c to the site of oxidation
(Table IB), thus showing that these structures of mitochondrial origin had no outer membrane integrity.
In situ observations of glucose-starved root tips (Fig. 3c)
showed that the cells corresponding to the meristematic
region of nonstarved roots were no longer meristematic, and
presented the structural organization of more differentiated
cells, with extended vacuolization, development of the en-
domembrane system, and maturation of organelles. Mitochondria, although smaller than mitochondria of nonstarved
differentiated cells, were more dense and contained distinct
cristae protruding from the inner membrane. This effect could
be ascribed to glucose starvation because the terminal cells
of excised root tips that had been incubated for 48 h in the
presence of 0.2 M glucose remained meristematic (data not
shown).
Structural and Functional Characteristics of HigherDensity Mitochondria in Glucose-Starved Maize Root Tips
In contrast with LB preparations, HB preparations from
starved material, which corresponded to fractions 1-2 of the
Table II. Respiratory Activity of Maize Root Tip Mitochondria prior to Glucose Starvation (A) and after
48 h of Glucose Starvation (B)
The results are the mean (±SE) of three to five measurements from at least two experiments, except
in the case of the respiration of 2-oxoglutarate by nonstarved LB mitochondria (A) and of pyruvate
by glucose-starved LB preparations (B), where only one experiment was carried out.
HB Mitochondria
LB Mitochondria
Substrate
RState 3 respiration
espiratory ADP/O
control
nmoIO2. min '. mg-'
Respiration in the
Respiratory
presence of ADP
nmoIO2. min '. mg-'
control
A.
Succinate
NADH
44 ± 7
2-Oxoglutarate
Pyruvate
84 ± 8
14 ± 2
42 ± 6
Citrate
12 ± 1
1.9
2.7
2.0
2.1
2.0
Succinate
NADH
60 ± 3
91 ± 6
13 ± 1
32 ± 2
3.7±0.7
2
3
2.5
2.5
1.6
1.1
1
2
1.8
2.3
13 ± 1
18 ± 3
6.2
9± 1
4± 1
1
1.5
1
1
1.3
1.3
1
1.3
1.4
1.4
26 ± 8
Not determined
Not detected
1.5
1.4
B.
2-Oxoglutarate
Pyruvate
Citrate
1± 1
1
1
EFFECTS OF GLUCOSE STARVATION ON ROOT TIP MITOCHONDRIA
1 895
Figure 2. Electron micrographs of Percoll-purified mitochondria from nonstarved maize root tips. LB (a and b) and HB (c and d) preparations
were obtained as described in Figure 1A and analyzed by EM. Low magnification (a, x1 7,300; c, x1 7,500) and high magnification (b, X50,200;
d, x43,300) views are given. A large majority of organelles were recognized as mitochondria. Some membrane vesicles could be observed.
1896
Figure 3. Electron micrographs of the meristematic and submeristematic regions of maize
root prior to and after glucose starvation. Cells
in the meristematic region (a) and the submeristematic, differentiated region (b) of 3-mm
long excised maize root tips prior to glucose
starvation, and cells in the terminal region of
excised maize root tips that had been subjected
to 48 h of glucose starvation (c) were observed
with a x27,400 magnification. The views show
the ultrastructure of mitochondria in these
cells. The cells in the terminal region of glucosestarved root tips were no longer meristematic
and presented the structural organization of
more differentiated cells.
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EFFECTS OF GLUCOSE STARVATION ON ROOT TIP MITOCHONDRIA
Table Ill11. Specific Activities of Mitochondrial Matrix and Inner
Membrane Enzymes in Mitochondria Isolated from Maize Root
Tips prior to Glucose Starvation (A) or after 48 h of Glucose
Starvation fB)
The specific activities are expressed in nmol.minw1.mg-'. The
results are the mean (±SE) of at least three experiments.
LB Preparation
HB Preparation
A.
NAD-IDH
NAD-GDH
SDH
Fumarase
18 ±9
82 ±54
33 ±2
330 ± 85
8± 2
54 ±40
23± 2
126 ± 23
NAD-IDH
NAD-GDH
SDH
Fumarase
37 ±4
129 ±43
27 ±8
191 ± 11
1.3 ±0.1
23 ±11
2.2 ±0.8
23 ± 15
B.
gradient (Fig. B), were enriched in mitochondrial
(Table IB) and showed significant respiration
with strong respiratory control (Table IIB). EM (Fig. 4)
Percoll
enzyme markers
rates
showed that these mitochondria
were
to the mature mitochondria isolated
ultrastructurally
similar
tips (Fig. 2). However, some differences could be obHigher-density mitochondria from glucose-starved
maize root tips were more dense (1.070) than nonstarved
mitochondria (Fig. 1) and showed a higher specific activity
for IDH-NAD (Table IIIB) and a higher respiration rate for
succinate (Table IIB). As discussed above, higher-density,
served.
more
differentiated mitochondria could be observed in situ
throughout
the root
tip (Fig. 3).
Decrease of the Total Mitochondrial Pool under the
Effects of Glucose Starvation
The yields of protein and mitochondrial enzyme activities
during mitochondria isolation are shown in Table IV. Values
of yield above 100% in the crude extract were probably due
to differences of enzyme stability in the different extraction
mediums used in
Brouquisse
extraction
et
was
al.
present work and
the
(4).
In
this latter work,
carried out in the presence of
in
a
a
the
work of
more
drastic
detergent
and
of protease inhibitors. The values thus obtained could be
taken
as a
reference to
control and
assess
glucose-starved
yield of mitochondria from
tips. Although the efficiency
the
root
protein extraction was lower in 48-h starved maize root
tips, the extraction procedure gave comparable yields of
of
fumarase and NAD-IDH in the crude extracts obtained from
nonstarved and
glucose-star'ved
maize root
tips
for the iso-
lation of mitochondria.
Furthermore, in both
the latency of NAD-IDH
(Table IV) and throughout
cases
the crude extract
high
subsequent steps
in
compare the amounts of mitochondrial proteins obtained
from control and glucose-starved maize root tips. Thus, the
amount of protein in the Percoll-purified organellar fraction
from 1000 maize root tips decreased from 9.5 mg (±1.5 SE)
to 6 mg (±1 SE) after 48 h of glucose starvation.
The size of the mitochondrial pool was also estimated from
the comparison of specific activities in LB and HB preparations with total activities in the tissue as determined by
Brouquisse et al. (4). In the case of nonstarved maize root
tips, calculations using the specific activities of HB or LB
preparations underestimated and overestimated, respectively,
the amount of mitochondrial protein (Table V). These different estimations indicated that the amount of mitochondrial
protein per root tip decreased during the first 48 h of glucose
starvation. Thus, mitochondrial proteins appeared to be degraded, which would be in accordance with previous studies
(4, 15). However, the observed decrease did not seem to
affect the pool of higher-density mitochondria. The amount
of protein in the Percoll-purified HB preparations from 1000
maize root tips varied from 2.5 mg (±0.5 SE) to 4 mg (±1 SE)
after 48 h of glucose starvation.
DISCUSSION
from nonstarved maize
root
was
the
procedure (Table I and other data
not shown), thus indicating that mitochondria were not significantly damaged during extraction and isolation. The subof the
sequent steps for the isolation of mitochondria gave similar
yields of mitochondrial enzyme activities whether prior to or
after glucose starvation treatment (Table IV). As the behavior
of the material during the isolation procedure was comparable prior to and after glucose starvation, it was possible to
19
1897
The mitochondria isolated from maize root tips were found
heterogeneous with respect to density, ultrastructure,
and respiratory properties. The respiratory control and
ADP/O ratios of higher-density mitochondria, although low,
were of the same order of magnitude as those reported for
mitochondria from other root tissues (1 3). The low respiration
rates and the weak respiratory controls shown in vitro by
low-density mitochondria may have been the result of damage during isolation. However, these characteristics were
consistent with in situ observations showing immature mitochondria with a clear matrix and no cristae. Furthermore,
no significant leakage of matrix material occurred during
isolation, since the latency of NAD-IDH was high throughout
the procedure. Thus, the observed heterogeneity was not
artifactual and seemed to be correlated, in accordance with
earlier studies (5), with the existence of mitochondria at
various stages of maturation in m'eristematic and differentiating root tissues.
Glucose starvation has immediate drastic effects on respiration (4, 18). This decrease of respiration has been shown to
result essentially from a decrease in the levels of respiratory
substrates and of ADP (4). In maize root tips, over 60% of
the initial respiration was thus rapidly recovered after transferring glucose-starved maize roots to a medium containing
0.2 m glucose (4). The present work shows that a significant
pool of functional mitochondria was maintained in glucosestarved root tips and could sustain this rapid recovery of
respiration. However, after 6 h of glucose starvation, the
complete recovery is biphasic and the second phase leading
to 100% recovery requires approximately 10 h (4), thus
showing that long-term readjustments are also necessary.
Indeed, the present work shows that, after 48 h of glucose
starvation, there was a decrease of the total mitochondrial
pool when expressed as the amount of mitochondrial protein
per tip. The analysis on Percoll gradients and in vitro EM
studies showed that lower- and intermediate-density mitoto be
1898
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COUEE ET AL.
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Plant Physiol. Vol. 100, 1992
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Figure 4. Electron micrographs of Percoll-purified mitochondria from 48-h glucose-starved maize root tips. LB (a and b) and HB (c and d)
preparations were obtained as described in Figure 1 B and analyzed by EM. Low magnification (a, x 16,900; c, x 1 7,500) and high magnification
(b, x43,900; d, X43,300) views are given. In HB preparations, a large majority of organelles were recognized as mitochondria, whereas some
membrane vesicles could be observed. In LB preparations, a large majority of various membrane vesicles with an electron-transparent
content were observed. Views a and b show vesicles of mitochondrial origin with inner cristae distinctly attached.
EFFECTS OF GLUCOSE STARVATION ON ROOT TIP MITOCHONDRIA
Table IV. Mitochondrial Integrity and Yields of Protein and Enzyme
Activities in the Course of Mitochondria Isolation from 1000 Maize
Root Tips prior to and after 48 h of Glucose Starvation
The yields (±SE) in the different steps of mitochondria isolation
obtained as described in "Materials and Methods" were calculated
from the means (±SE) of at least three experiments, relative to the
total tissue contents, as determined by Brouquisse et al. (4). The
protein contents were expressed in or, in the case of the values
given by Brouquisse et al. (4), converted into mg equivalent of 'yglobulin. In the case of latency values, only two results are given.
Glucose
After 48 h of
Glucose
Starvation
Starvation
Prior to
Total tissue content (4)
Protein (mg equivalent of y-globulin)
Fumarase activity (gmol-min-')
NAD-IDH activity (Mmol-min-')
Crude extract
Yield of proteins (%)
Yield of fumarase activity (%)
Yield of NAD-IDH (%)
Latency of NAD-IDH (%)
Crude mitochondria
Yield of fumarase activity (%)
Yield of NAD-IDH activity (%)
Percoll gradient
Yield of fumarase activity (%)
540 ± 40 300 ± 30
7.0 ± 0.2 4.0 ± 0.2
0.6 ± 0.1 0.30 ± 0.07
50 ± 10
150 ± 50
80 ± 30
90-99
31 ± 5
100 ± 10
190 ± 50
96-97
40 ± 10
30 ± 10
28 ± 3
40 ± 10
27 ± 5
21 ± 5
chondria could no longer be detected, in contrast with higherdensity mitochondria, which were maintained with the same
respiratory properties as nonstarved mitochondria. This disappearance of lower-density mitochondria was confirmed by
in situ EM studies showing the disappearance of immature
mitochondria (Fig. 3).
This selective disappearance may suggest that degradation
processes had affected these mitochondria, which would be
consistent with the degradation of the total lipid and protein
pools (4) and the decrease of the cardiolipin pool (15). However, in situ EM studies of the terminal regions of the root
tips after glucose starvation (Fig. 3) indicated that differentiation was not inhibited during glucose starvation, because
higher-density mitochondria with more developed cristae
were observed instead of the lower-density, immature mitochondria that were observed prior to glucose starvation.
However, these mitochondria differentiating during glucose
starvation may show significant differences, such as in the
development of cristae (Fig. 3), from the mitochondria present
in differentiated cells prior to glucose starvation. Furthermore, EM observations in the present work and the inhibition
of elongation (4) have shown that mitotic activity was inhibited during glucose starvation. Thus, the disappearance of
lower- and intermediate-density mitochondria would be the
consequence of continued cellular differentiation and of the
inhibition of the formation of immature mitochondria
through mitotic activity. In parallel, these processes would
result in the production of more mature mitochondria.
However, these effects on mitotic activity and differentiation do not preclude the existence of degradation processes
as suggested by the decrease of the total mitochondrial pool.
The membrane vesicles that were observed in LB preparations
after glucose starvation were at least partially of mitochondrial origin based on ultrastructural and respiratory characteristics (Fig. 4, Table IIB). No cognate structures were
observed in situ. Thus, these vesicles observed in isolated
preparations may have been formed during the isolation
process, probably because of the increased fragility of the
glucose-starved material, because no such structures were
obtained from nonstarved root tips. This increased fragility
could affect all types of mitochondria, whatever their degree
of differentiation. Indeed, at later stages of glucose starvation,
the replenishment of respiratory substrates with 0.2 M glucose
does not induce any rapid increase of respiration (4), which
would suggest that at that stage functional mitochondria are
also degraded.
Thus, in glucose-starved maize root tips, the interactions
between differentiation and degradation processes result in
the depletion of the pool of less differentiated mitochondria
and the conservation of a significant pool of more mature
Table V. Estimation of the Mitochondrial Pool Size in 1000 Maize Root Tips prior to and after 48 h of
Glucose Starvation
The mitochondrial pool size was expressed as the quantity of mitochondrial protein in 1000 maize
root tips. This was calculated as the ratio (±SE) of total mitochondrial enzyme activities (Mmol -min-')
in the tissue (4) to the specific activities (Mmol min-1. mg 1) of these enzymes in Percoll-purified
mitochondria (Table l1l). In the case of nonstarved root tips, where two populations of mitochondria
were isolated, the calculations using the specific activities in LB and HB mitochondria gave an upper
limit and a lower limit, respectively, to the estimated amount of mitochondrial protein. In the case
of starved root tips, where only one population of mitochondria was isolated, only one estimation
was calculated.
Estimated Amount of Mitochondrial Protein
Prior to glucose starvation
Lower limit
Upper limit
20 ± 4
30 ± 15
50 ± 10
50 ± 5
60 ± 20
67 ± 12
After 48 h of
glucose starvation
mg
Estimation based on fumarase activity
Estimation based on NAD-IDH activity
Estimation based on SDH activity
1 899
25 ± 2
8±3
10 ± 5
1 900
COU EE ET AL.
mitochondria at least during the first stages of the starvation
treatment. Current work is in progress to assess the participation of functional mitochondria in the metabolic response
of the tissue to glucose starvation and the molecular basis of
the degradation processes.
1.
2.
3.
4.
5.
6.
7.
8.
9.
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