Copyright © Physiologia Plantarum 2011, ISSN 0031-9317
Physiologia Plantarum 144: 20–34. 2012
The role of recovery of mitochondrial structure and function
in desiccation tolerance of pea seeds
Wei-Qing Wanga , Hong-Yan Chenga , Ian M. Møllera,b and Song-Quan Songa,∗
a
b
Group of Seed Physiology and Biotechnology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China
Department of Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark
Correspondence
*Corresponding author,
e-mail: sqsong@ibcas.ac.cn
Received 25 April 2011;
revised 3 July 2011
doi:10.1111/j.1399-3054.2011.01518.x
Mitochondrial repair is of fundamental importance for seed germination.
When mature orthodox seeds are imbibed and germinated, they lose their
desiccation tolerance in parallel. To gain a better understanding of this
process, we studied the recovery of mitochondrial structure and function in
pea (Pisum sativum cv. Jizhuang) seeds with different tolerance to desiccation.
Mitochondria were isolated and purified from the embryo axes of control
and imbibed–dehydrated pea seeds after (re-)imbibition for various times.
Recovery of mitochondrial structure and function occurred both in control
and imbibed–dehydrated seed embryo axes, but at different rates and to
different maximum levels. The integrity of the outer mitochondrial membrane
reached 96% in all treatments. However, only the seeds imbibed for 12 h
and then dehydrated recovered the integrity of the inner mitochondrial
membrane (IMM) and State 3 (respiratory state in which substrate and ADP
are present) respiration (with NADH and succinate as substrate) to the control
level after re-imbibition. With increasing imbibition time, the degree to
which each parameter recovered decreased in parallel with the decrease
in desiccation tolerance. The tolerance of imbibed seeds to desiccation
increased and decreased when imbibed in CaCl2 and methylviologen
solution, respectively, and the recovery of the IMM integrity similarly
improved and weakened in these two treatments, respectively. Survival of
seeds after imbibition–dehydration linearly increased with the increase in
ability to recover the integrity of IMM and State 3 respiration, which indicates
that recovery of mitochondrial structure and function during germination has
an important role in seed desiccation tolerance.
Introduction
Desiccation tolerance generally refers to the ability of
organisms to survive loss of 80 to 90% of protoplasmic
water to reach <0.3 g water g−1 dry matter. Under these
conditions, there is no free water in the living cells
and the structure of macromolecules and membranes is
protected by specific mechanisms (Hoekstra et al. 2001,
Oliver et al. 2000). This phenomenon is widespread
across the plant kingdom, including ferns, mosses,
pollen, angiosperm resurrection plants and orthodox
seeds. In orthodox seeds, desiccation tolerance is
acquired during seed maturation approximately halfway
through development. This trait ensures that seeds pass
unharmed through maturation drying and retain viability
in the dry state for long periods of time under natural
Abbreviations – AOX, alternative oxidase; BSA, bovine serum albumin; CCO, cytochrome c oxidase; DTT, dithiothreitol;
FCCP, p-trifluoromethoxyphenylhydrazone; IMM, inner mitochondrial membrane; MOPS, 3-(N-morpholino)propanesulphonic
acid; MV, methylviologen; OMM, outer mitochondrial membrane; RCR, respiratory control ratio (State 3 rate/State 4 rate);
ROS, reactive oxygen species; SHAM, salicylhydroxamic acid; State 3, respiratory state in which substrate and ADP are
present; State 4, respiratory state in which all ADP has been converted into ATP; TX, Triton X-100.
20
Physiol. Plant. 144, 2012
conditions (Kermode and Finch-Savage 2002, Vertucci
and Farrant 1995).
Bewley (1979) suggested that three protoplasmic properties are required for plant or plant tissue to establish
desiccation tolerance: (1) minimize damage from desiccation and/or rehydration, (2) maintain cellular integrity
in the dry state and (3) mobilize repair mechanisms
on rehydration. These criteria highlight the importance
of two basic cellular process, protection and repair,
in desiccation tolerance (Oliver et al. 2005). Many
specific protective mechanisms have been implicated
in acquisition and maintenance of seed desiccation
tolerance, including reduction of the degree of vacuolization, intracellular de-differentiation, ‘switching off’
metabolism, accumulation of protective molecules, such
as late embryogenesis abundant (LEA) proteins, sucrose
and certain oligosaccharides, as well as the presence
and efficient operation of antioxidant systems (Berjak
and Pammenter 2008, Pammenter and Berjak 1999).
Although repair mechanisms have been identified in
mosses and resurrection plants (Oliver 1996, Oliver et al.
2005, Proctor et al. 2007, Rascio and La Rocca 2005),
their nature is still unclear in seed desiccation tolerance.
In seeds, the mitochondrion is the major organelle
for energy supply and its function is tightly coupled
to seed germination (Bewley 1997). During maturation drying, seed mitochondria disassemble because of
desiccation stress and in electron micrographs of dry
seeds the mitochondria appear with poorly differentiated structure (Ehrenshaft and Brambl 1990, Logan et al.
2001, Morohashi et al. 1981). A process of repair of
mitochondrial structure and function is necessary for
seed germination during the imbibition process (Bewley
1997). Many studies have shown that mitochondria in
dry seeds recover the mature structure with well-defined
cristae and a dense matrix during the germination process (Benamar et al. 2003, Ehrenshaft and Brambl 1990,
Howell et al. 2006, Logan et al. 2001, Morohashi et al.
1981). These resurrected mitochondria then supply the
energy and metabolic intermediates that seed germination requires.
Mitochondria are one of the targets for various stress
damage, probably because of their large turnover of
reactive oxygen species (ROS; Amirsadeghi et al. 2007,
Macherel et al. 2007, Møller 2001, Møller et al. 2007,
Pastore et al. 2007). Prasad et al. (1994) indicated that
the ability of maize seedlings to recover from chilling
injury was, at least in part, because of the recovery of
mitochondrial function. Severe seawater stress decreases
durum wheat germination and delays seedling growth,
and damages the mitochondrial succinate-dependent
oxidative phosphorylation at the same time (Flagella
et al. 2006). It is possible that loss of desiccation
Physiol. Plant. 144, 2012
tolerance in seeds is because of desiccation damage
to their mitochondria. Indeed, the respiration rate and
activity of cytochrome c oxidase (CCO) and malate
dehydrogenase in mitochondria from axes of recalcitrant
Antiaris toxicaria seeds and from embryos of orthodox
maize seeds all decreased during dehydration (Song
et al. 2009, Wu et al. 2009), but we do not have any
information about their recovery during germination. In
view of the importance of mitochondrial repair for seed
germination, we here hypothesize that this process is
also necessary for seed desiccation tolerance.
When mature seeds are imbibed and then dehydrated,
survival decreases gradually with increasing imbibition
time, that is, desiccation tolerance is lost during
seed germination (Dasgupta et al. 1982, Koster and
Leopold 1988, Reisdorph and Koster 1999, Senaratna
and McKersie 1983). Thus germinating orthodox seeds
resemble recalcitrant seeds, and this system has therefore
been suggested to be a useful model for understanding
the mechanisms of recalcitrance (Sun 1999). In nature,
seeds may experience several imbibition–desiccation
cycles during germination because of fluctuations in
soil moisture. After the point at which the seeds
have lost their desiccation tolerance, these cycles will
decrease seed germination and seedling formation. Thus
understanding desiccation tolerance will be important
for optimizing not only storage of recalcitrant seeds, but
also crop yield.
Desiccation tolerance can be improved in germinating
seeds by treatment with certain exogenous chemicals,
such as polyethylene glycol or abscisic acid (Bruggink
and Vandertoorn 1995, Buitink et al. 2003). This method
can be very useful for studying the mechanism of
desiccation tolerance (Faria 2006). Production of ROS
during dehydration is considered as one of the reasons
causing desiccation damage (Bailly 2004). Thus the
application of a ROS producer, such as methylviologen
(MV) to desiccation-tolerant seeds would be expected
to decrease or remove the tolerance. Ca2+ acting as
a second messenger can improve the response of
plant to many adverse stresses (Kudla et al. 2010,
Lecourieux et al. 2006, Sanders et al. 2002). Ca2+ is
also important for membrane structure (Møller 1983,
White and Broadley 2003), but little is known about its
effect on desiccation tolerance.
In this study, we have investigated the change of desiccation tolerance of pea (Pisum sativum cv. Jizhuang)
seeds imbibed in distilled water for various times,
and the effect of CaCl2 and MV, a producer of ROS
(Halliwell and Gutteridge 2007) on desiccation tolerance of germinating seeds. Mitochondria were isolated
and purified from the embryo axes of the untreated
seeds and of a series of imbibed–dehydrated seeds
21
(tolerant, partly tolerant and intolerant to desiccation)
and used to monitor the recovery of mitochondrial structure and function during seed (re-)imbibition. A normal
cumulative equation was applied to fit the time courses
of mitochondrial parameters during (re-)imbibition. Our
results indicated that the recovery of mitochondrial structure and function during germination is important in seed
desiccation tolerance.
Materials and methods
Plant material
Pea (P. sativum cv. Jizhuang) seeds were obtained from
Guangling Seed Company (Datong, Shanxi, China) in
September 2008 and were kept in plastic bags at 5◦ C
until used. The water content of the seeds was about
0.10 g H2 O g−1 dry weight.
Seed imbibition, dehydration and survival
Seed imbibition and germination
Three replicates of 25 pea seeds each were placed on
two layers of filter paper and imbibed in 40 ml distilled
water or treatment solution (20 mM CaCl2 or 2 mM
MV both dissolved in distilled water) in closed 15-cm
diameter Petri dishes at 20◦ C in darkness for up to 48 h.
Radicle protrusion of 2 mm was used as the criterion of
germination, and the number of germinated seeds was
counted regularly throughout the test period.
Another sample of three replicates of 25 pea seeds
each was imbibed in distilled water or treatment solution (20 mM CaCl2 or 2 mM MV) to determine seed
water uptake. These seeds were regularly removed from
the Petri dishes to determine the seed weight, which was
used to calculate the seed water content.
Dehydration and survival
The pea seeds were imbibed in distilled water or
treatment solution (20 mM CaCl2 or 2 mM MV) at 20◦ C
in darkness for 12–48 h, and then dehydrated to a water
content of about 0.10 g g−1 by burying them in activated
silica gel [1:8 (v:v) seeds:silica gel] within a closed plastic
box for 24–36 h.
The untreated (control) and imbibed–dehydrated
seeds were germinated on two layers of filter paper
moistened with 40 ml of distilled water in Petri dishes
(15-cm diameter) at 20◦ C in darkness for 7 days to
measure seed survival. Likewise, seeds imbibed in CaCl2
or MV for 25 h were tested for germination and survival.
Seeds showing a normal epicotyl and primary radical
were considered as being alive.
The control seeds (CK) imbibed in distilled water for
4, 12, 18, 24, 30 and 36 h, and the seeds imbibed in
distilled water for 12, 25 or 36 h and dehydrated (H12D,
H25D or H36D), as well as seeds imbibed in CaCl2 or
MV for 25 h and dehydrated (Ca25D or MV25D) were
re-imbibed in distilled water for 4, 8, 12, 18, 24 and
30 h and then used for the isolation and purification of
mitochondria (Fig. 1)
Determination of water content
The water content of seeds was determined according
to the method of International Seed Testing Association
(1999) and expressed on a dry mass basis (g H2 O g−1
dry weight).
Transmission electron microscopy
Embryo axes of CK, H12D and H36D (labels see
Fig. 1) seeds were used for fixation directly or after
(re-)imbibition of the seeds for 24 h, respectively.
Control seeds (CK)
No imbibition
Imbibition for 12 h
Imbibition for 25 h in
distilled water CaCl2
No dehydration
(CK)
Imbibition for
4, 12, 18, 24, 30, 36 h
Dehydration
(H12D)
(H25D
Imbibition for 36 h
MV
Dehydration
Ca25D
MV25D)
Dehydration
(H36D)
Re-imbibition for
4, 8, 12, 18, 24, 30 h
Excision of embryo axis and purification of mitochondria
Fig. 1. Protocol used for the imbibition, dehydration and subsequent re-imbibition of pea seeds. CK, H12D, H25D, Ca25D, MV25D and H36D are
dry seeds with a water content of about 0.10 g g−1 .
22
Physiol. Plant. 144, 2012
Mitochondria isolated and purified from embryo axes
of the seeds (re-)imbibed for 24 h were also used. The
embryo axes and purified mitochondria were fixed in
2.5% (v/v) glutaraldehyde, 100 mM sodium phosphate
(pH 7.2) (for purified mitochondria, the solution also contained 0.4 M sucrose) for 2 h. The fixed embryo axes that
had been rinsed (twice) and purified mitochondria that
had been centrifuged (10 000 g for 10 min) and rinsed
(twice) were post-fixed in 1% (v/v) osmium tetroxide,
100 mM sodium phosphate (pH 7.2) for 2 h, dehydrated
in an ethanol series and then embedded in Epon resin.
Ultra-thin sections were cut and stained with uranyl
acetate followed by lead citrate before observation. The
electron micrographs were recorded with a JEM-1230
transmission electron microscope (JEOL, Tokyo, Japan).
Isolation and purification of mitochondria
Mitochondria were prepared according to the method of
Benamar et al. (2003) modified as follows: 200 embryo
axes were homogenized using a precooled mortar and
pestle in 20 ml of extraction buffer composed of 0.4 M
sucrose, 20 mM 3-(N -morpholino)propanesulfonic acid
(MOPS), 1 mM EDTA, 4 mM cysteine, 10 mM MgCl2 ,
0.4% (w/v) bovine serum albumin (BSA) and 0.5% (w/v)
insoluble polyvinylpyrrolidone, adjusted to pH 7.5 with
KOH. After washing the mortar with 20 ml extraction
buffer, the total 40 ml homogenate was filtered through
four layers of gauze. The filtrate was centrifuged at 100 g
for 5 min and 500 g for 10 min, and the supernatant was
then centrifuged at 15 000 g for 20 min. The resulting
pellet was suspended in washing buffer composed of
0.4 M mannitol, 1 mM EDTA, 0.2% (w/v) BSA, 10 mM
MOPS–KOH, pH 7.2 and layered on to a discontinuous
Percoll gradient: 3 ml of 25% Percoll containing 0.4 M
mannitol, 5 mM MgCl2 , 0.2% BSA, 10 mM MOPS-KOH,
pH 7.2, on top of 3 ml of 35% Percoll containing 0.4 M
sucrose, 5 mM MgCl2 , 0.2% BSA, 10 mM MOPS-KOH,
pH 7.2. After centrifugation at 10 000 g for 20 min, the
mitochondrial band at the interface between 25 and 35%
Percoll solution was collected and diluted eight times
with washing buffer (without BSA), and then pelleted at
13 000 g for 15 min to remove the Percoll. This dilution
and pelletation was repeated once. The pellets were
finally resuspended in washing buffer (without BSA) and
used for assay of enzyme activity and determination of
oxygen consumption and protein content. All procedures
were carried out at 4◦ C.
oxidation of reduced cytochrome c at 550 nm. The
latency of CCO activity was calculated as described by
Møller et al. (1987).
Latency (%) = 100 × [(rate + TX) − (rate − TX)]/
(rate + TX),
(1)
where (rate + TX) and (rate − TX) are the oxidation
rate of reduced cytochrome c by CCO in the reaction
medium with and without 0.05% (w/v) Triton X-100
(TX), respectively.
Measurements of oxygen consumption
Oxygen consumption of mitochondria was measured
using an oxygen electrode system (Oxytherm, Hansatech
Instruments, Pentney, King’s Lynn, Norfolk, UK). The
reaction medium contained 0.4 M mannitol, 10 mM
KH2 PO4 , 10 mM KCl, 5 mM MgCl2 and 0.1% (w/v)
BSA, 10 mM MOPS-KOH, pH 7.5, in a total volume
of 1 ml. For substrate oxidation, first mitochondria
(80–150 μg protein) and the substrate (1.5 mM NADH
or 10 mM succinate) was added followed by two
additions of ADP, the first time (50 μM) to activate
and the second time (100 μM) to obtain States 3
and 4 respiration and to give the ADP/O ratio. A
1 mM EGTA was added before NADH in some control
experiments to remove Ca2+ from the membranes,
which might be sufficient to give maximal NADH
oxidation activity (Møller 1983, Møller et al. 1981,
Nash and Wiskich 1983). Uncoupler, carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP; 2 μM) was
used to uncouple NADH oxidation to obtain another
measure of uninhibited respiration rate. For estimation
of alternative oxidase (AOX) capacity, the cytochrome
oxidase pathway during NADH oxidation was inhibited
by potassium cyanide (1 mM), pyruvate (10 mM) and
dithiothreitol (DTT; 10 mM) were added to activate
AOX, and finally salicylhydroxamic acid (SHAM; 2 mM)
was added to inhibit AOX. AOX capacity was calculated
as the rate after addition of DTT and pyruvate minus
the rate after SHAM addition (= residual respiration).
The mitochondrial respiration was expressed as nmol
O2 mg protein−1 min−1 .
Determination of protein
The concentration of protein in the purified mitochondria
was determined according to Bradford (1976), using BSA
as a standard.
Assay of CCO activity
Statistical analysis
Activity of CCO (EC 1.11.1.5) was assayed using the
method of Møller et al. (1987), which measures the
A modified population based model (Eqn 2) was applied
to fit the time courses of recovery of mitochondrial
Physiol. Plant. 144, 2012
23
A
B
C
D
Fig. 2. Water uptake, time course of germination and survival of pea seeds. (A) Water content of seeds imbibed for 0–48 h in distilled water (H2 O),
CaCl2 and MV. (B) Germination of seeds imbibed for 0–48 h in H2 O, CaCl2 and MV. (C) Survival of seeds after imbibition and dehydration. Seeds
imbibed in H2 O, CaCl2 and MV for 12–48 h, dehydrated in silica gel to water content of about 0.1 g g−1 , and then germinated in two layers of
moist filter paper at 20◦ C for 10 days to detect survival. (D) The survival of seeds before and after dehydration. Seeds imbibed in H2 O, MV or CaCl2
for 25 h were directly germinated in distilled water (solid bar) or were dehydrated first and then germinated in distilled water (open bar) for 10 days
to detect the survival. The data are means with standard error of mean of three replicates of 25 seeds each.
structure and function. This model, derived from the
model describing seed germination and dormancy
release (Bradford 2002, Wang et al. 2009), assumes that
recovery of the mitochondrial structure and function
during (re-)imbibition follows a cumulative normal
distribution:
y = ymax × [(x − μ)/σ ]
(2)
where y is a parameter reflecting mitochondrial structure
and function at a given imbibition or re-imbibition time
x, ymax is the maximum value of y, is the normal
probability integral and μ and σ are mean and standard
deviation of original distribution of y.
GRAPHPAD PRISM 5.0 (GRAPHPAD Software, San Diego,
CA) was applied for nonlinear regression. F test with
PRISM 5.0 was used to test whether data sets of different
treatments could be shared with a global curve (Motulsky
and Christopoulos 2003).
24
Results
Water uptake, germination and desiccation
tolerance during imbibition
Water uptake of pea seeds showed a triphasic behavior
with a rapid initial water uptake at the first 10 h, followed
by a slower uptake between 10 and 30 h and finally a
slightly faster phase after 30 h (Fig. 2A). Water uptake
in distilled water, Ca2+ solution and MV solution was
the same for the first 15 h and at 25 h, when seeds were
dried, the water content differed by only 0.05 g g−1
between the three treatments (results not shown).
Seeds began to germinate at 16 h, and reached
maximum germination (>95%) at about 36 h when
imbibed in distilled water (Fig. 2B). Germination was
delayed in the presence of CaCl2 (important for
structure) and MV (causes ROS formation), and the final
germination percentage was also reduced by 10% in the
presence of MV in the imbibition medium (Fig. 2B).
Physiol. Plant. 144, 2012
A
B
C
D
E
F
G
H
I
Fig. 3. Transmission electron micrographs of mitochondria and cells in pea seed embryo axis (A, B, D, E, G and H) and purified mitochondria from
pea seed embryo axis (C, F and I). The control seeds and seeds imbibed in distilled water for 12 and 36 h and dehydrated (H12D and H36D) were
not (re-)imbibed (‘dry embryo axis’, A, D and G) or (re-)imbibed for 24 h [‘(re-)imbibed embryo axis’, B, E and H], respectively, and the embryo axes
were excised and fixed. A, B, D, E, G and H show transmission electron micrographs of mitochondria in embryo axes cell, and the insert figures are
transmission electron micrographs of embryo axis cells at lower magnification. The embryo axes of seeds re-imbibed for 24 h were also excised and
used for purification and fixation of mitochondria (purified mitochondria, C, F and I). The scale bar in A represents 0.5 μm and applies also to B, D,
E, G and H. The scale bar in A insert represents 5 μm, and applies also to inserts in B, D, E, G and H insert. The scale bar in C represents 0.2 μm
and applies also to F and I. The water contents of CK, H12D and H36D seed embryo axes (re-)imbibed for 24 h were 3.47, 3.43 and 4.30 g g−1 ,
respectively (B, E and H). M, mitochondrion.
When the imbibed pea seeds were dehydrated,
they maintained their maximum viability up to 15 h
of imbibition, after which seed death occurred in
a log cumulative normal distribution over imbibition
time (Fig. 2C). No seeds survived after imbibition for
48 h followed by dehydration (Fig. 2C). At the same
imbibition time followed by dehydration, the presence
of CaCl2 in the imbibition solution increased, and MV
decreased, the survival percentage of seeds, as indicated
by the shift in the T50 value (Fig. 2C). Imbibition in
CaCl2 and MV for 25 h did not in itself affect seed
viability, as seeds imbibed in these two solutions and
then germinated without an intervening dehydration all
survived (Fig. 2D).
Physiol. Plant. 144, 2012
Transmission electron micrographs
of mitochondria
A morphological examination of mitochondria in situ
and after isolation was performed using transmission
electron microscopy (Fig. 3). In the control seeds (CK,
Fig. 3A) and imbibed–dehydrated seeds (H12D and
H36D, Fig. 3D, G), mitochondria had little discernible
internal structure. After 24 h of re-imbibition, the
mitochondrial structure was repaired, but only CK and
H12D had fully differentiated mitochondria (Fig. 3B,
C, E, F) and in H36D seeds the mitochondria could
not recover the structure with well-defined cristae
(Fig. 3H, I). The embryo axis cells in H36D seeds
became plasmolyzed after dehydration (Fig. 3G insert),
25
and the plasmolyzed cells were still plasmolyzed after
subsequent rehydration (Fig. 3H insert). This occurred
despite the fact that the embryo axes were fully imbibed
as seen from the high water content of the H36D axes
(Fig. 3H insert) in comparison with CK and H12D axes
after 24 h imbibition (Fig. 3B, E insert). Thus there may
be desiccation damage to the plasma membrane in the
H36D seeds.
Purification of embryo axis mitochondria
Mitochondria were isolated and purified from embryo
axes of germinating pea seeds [0–36 h (re-)imbibition]
pretreated in six different ways: control seeds (CK), seeds
imbibed in distilled water for 12, 25 or 36 h and then
dehydrated (H12D, H25D or H36D), or seeds imbibed
in CaCl2 or MV for 25 h and then dehydrated (Ca25D or
MV25D) (Fig. 1).
Mitochondrial purity was monitored in crude and
purified mitochondria by the specific activity of CCO,
an enzyme found only in the inner mitochondrial
membrane (IMM; Lardy and Ferguson 1969). The latency
of CCO activity was used to monitor the integrity of the
outer mitochondrial membrane (OMM), as an intact
outer membrane does not permit the CCO substrate,
reduced cytochrome c (12.5 kDa), to be oxidized by
CCO (Neuburger 1985).
The CCO specific activity increased 2.2 to 6.5fold from crude mitochondria to purified mitochondria
(results not shown). This was mainly because of a
large variation in the percentage of total CCO activity
in the crude mitochondria recovered in the purified
mitochondria. The recovery was linearly correlated with
OMM integrity (Fig. S1A, supporting information). OMM
integrity in purified mitochondria was linearly correlated
with that of crude mitochondria (Fig. S1B) and it did not
change as a result of purification (see x = y line in
Fig. S1B). In summary, when the OMM of the crude
mitochondria was relatively intact, a large percentage of
the mitochondria was recovered in the purified fraction.
Recovery of mitochondrial structure and function
CCO activity and integrity of OMM
CCO activity was low in embryo axis mitochondria from
CK seeds at 6 and 12 h of imbibition, but increased
rapidly thereafter (Fig. 4A). CCO activity was higher
in mitochondria from the imbibed–dehydrated seeds
H12D, H25D and H36D than in mitochondria from
CK seeds for the first 18 h of (re-)imbibition (Fig. 4A).
However, the maximum CCO activity to be recovered
was similar in CK, H12D and H25D seeds, and
significantly lower in H36D seeds (Fig. 4A). Compared
26
to H25D seeds, CaCl2 treatment (Ca25D) increased CCO
activity at all times of re-imbibition, while MV treatment
(MV25D) had no effect (Fig. 4B).
In CK mitochondria, CCO latency increased very
rapidly and reached 96% within 18 h of imbibition. In
imbibed–dehydrated seeds, the final CCO latency was
similar to that in CK seeds (Fig. 4C, D), whereas it took
longer to recover in H36D seeds (Fig. 4C). Pretreatment
of the seeds with CaCl2 or with MV (followed by
dehydration) had no effect on the recovery of CCO
latency during germination (Fig. 4D).
IMM integrity
The functional intactness of the IMM was estimated
by measuring the State 3 (respiratory state in which
substrate and ADP are present)/State 4 (respiratory state
in which all ADP has been converted into ATP) ratio,
the respiratory control ratio (RCR), as this ratio will be
relatively high when proton leakage across the IMM
is low. A similar parameter is uncoupled rate ratio
(FCCP/State 4).
Among seeds imbibed in H2 O, the recovery of IMM
integrity as indicated by RCR and the uncoupled rate
ratio, was more rapid in embryo axis mitochondria
from the H12D and H25D seeds than in the CK
mitochondria. However, only mitochondria from H12D
seeds could recover IMM integrity to the same high
level as the CK mitochondria (Fig. 5A, C). The degree of
IMM integrity after 36 h re-imbibition was lower for
H25D mitochondria and markedly lower for H36D
mitochondria (Fig. 5A, C). CaCl2 treatment (Ca25D)
promoted, while MV treatment (MV25D) weakened,
the recovery of the IMM integrity during re-imbibition
compared to H2 O treatment (H25D; Fig. 5B, D).
Respiration
A high respiratory activity per milligram mitochondrial
protein (relative to that of mitochondria purified from
pea leaves – Nash and Wiskich 1983) indicates that
the mitochondria are well able to contribute to seed
metabolism.
The addition of Ca2+ to mitochondria oxidizing
NADH in the standard medium did not change the
rate of oxygen consumption measured. However, in
the presence of EGTA the rate was reduced by 85%
indicating that external NADH oxidation in peed seed
mitochondria is dependent on Ca2+ (results not shown).
The NADH oxidation rates presented in Fig. 6A, B are
the maximum rates measured in the absence of EGTA.
State 3 respiration in CK mitochondria (NADH or
succinate as substrate) was very low at 4 h re-imbibition,
but increased rapidly to reach very high activities
Physiol. Plant. 144, 2012
A
B
C
D
Fig. 4. Recoveries of CCO activity and OMM integrity in purified mitochondria from pea embryo axes during seed (re-)imbibition. The seeds untreated
(CK), imbibed in distilled water for 12, 25 and 36 h and dehydrated (H12D, H25D and H36D; A and C) and in CaCl2 and MV for 25 h and dehydrated
(CaD25 and MVD25; B and D) were (re-)imbibed in distilled water for 4–36 h and the embryo axes of the seeds were excised and used for isolation and
purification of mitochondria. CCO activity of purified mitochondria was assayed (A and B) and the latency of the CCO activity (C and D) was calculated
to estimate the integrity of OMM. Shared regression curves were fitted to the CCO activity of H25D and MV25D (B; F3,30 = 1.708, P = 0.19), to the
latency of CCO activity of CK, H12D and H25D (C; F6,45 = 1.315, P = 0.27) and to the latency of CCO activity of H25D, Ca25D and MV25D (D;
F6,45 = 1.784, P = 0.12) seeds. The data are means ± standard error of mean (n = 3) and lines are regressed with Eqn 2.
after 36 h (Fig. 6A, C). In H12D, H25D and H36D
mitochondria, State 3 respiration was higher than the
respiration in control mitochondria at the start time, but
only H12D mitochondria reached the same maximum
level as the CK mitochondria after 30 h re-imbibition.
For H25D and H36D mitochondria, the rates after 30 h
were only 73 and 48% of CK mitochondria (NADH
as substrate; Fig. 6A) or 81 and 40% (succinate as
substrate; Fig. 6C) of CK mitochondria. State 3 respiration
recovered better in mitochondria from Ca25D seed
embryo axes than in mitochondria from H25D embryo
axes (Fig. 6B, D). Although the State 3 respiration of
MV25D mitochondria was a little higher than for H25D
mitochondria early during re-imbibition, the maximum
rate of respiration that the mitochondria could reach
was similar (Fig. 6B, D). Recovery of AOX capacity
Physiol. Plant. 144, 2012
was similar among CK, H12D and H36D mitochondria
(Fig. 6E), but more rapid and reaching higher activities in
H25D mitochondria (Fig. 6E). AOX capacity was higher
in Ca25D and MV25D mitochondria than in control
mitochondria (Fig. 6F). Generally, AOX capacity was
only a small part of the State 3 activity (Fig. S2), but
in CK mitochondria after 4 h re-imbibition they were
almost the same size (Fig. S2A).
We calculated the ADP/O ratio during NADH
oxidation and the values were all between 1.0 and
1.3 with no clear trend (results not shown).
Seed survival and mitochondrial recovery
According to the Eqn 2, the maximum integrity of
IMM and oxygen consumption to be recovered during
27
A
B
C
D
Fig. 5. Recovery of integrity of IMM in purified mitochondria from pea embryo axes during seed (re-)imbibition. Mitochondria were isolated and
purified from embryo axes of CK, H12D, H25D and H36D (A and C), and of CaD25 and MVD25 (B and D) after (re-)imbibition, respectively. Uncoupled
(FCCP), State 3 (St3) and State 4 (St4) rates of respiration were determined using NADH as substrate. The uncoupled rate ratio (FCCP/St4, A and B)
and RCR (St3/St4, C and D) were calculated to indicate the integrity of IMM. The data are means ± standard error of mean (n = 3) and lines are
regressed with Eqn 2.
(re-)imbibition was calculated and plotted against the
percentage of seed survival (Fig. 7). Seed survival
increased linearly with increase in the NADH- or
succinate-dependent State 3 respiration, uncoupled rate
ratio and RCR (Fig. 7A, B), whereas the pattern with AOX
capacity was not clear (Fig. 7A).
Discussion
Orthodox seeds acquire desiccation tolerance during
maturation and lose it during imbibition (Berjak and
Pammenter 2008, Kermode and Finch-Savage 2002). In
this, pea (P. sativum cv. Jizhuang) seeds gradually lost
their desiccation tolerance after imbibition for longer
than 15 h, and no seed survived (measured as the
ability to germinate within 7 days) after imbibition for
28
48 h followed by desiccation (Fig. 2C). The desiccation
tolerance of imbibed seeds was improved and impaired
when imbibition took place in CaCl2 and MV solutions,
respectively, instead of in distilled water (Fig. 2C).
When seeds were imbibed with CaCl2 , a small delay
in germination was found in comparison with seeds
imbibed in water. This may be because of the slight salt
stress caused by the CaCl2 solution.
One of the key functions of mitochondria is to
catalyze substrate oxidation and produce ATP for use
in cellular metabolism. Mitochondria are affected by
environmental stress, such as drought (Pastore et al.
2007), chilling (Prasad et al. 1994, Stupnikova et al.
2006), heavy metals like Cd2+ (Smiri et al. 2009)
and ageing (Benamar et al. 2003). In orthodox seeds,
mitochondria appear with poorly differentiated structure
Physiol. Plant. 144, 2012
A
B
C
D
E
F
Fig. 6. Respiration recovery in purified mitochondria from embryo axes during pea seed (re-)imbibition. Mitochondria were isolated and purified from
embryo axes of CK, H12D, H25D and H36D (A and C), and of CaD25 and MVD25 (B and D) seeds after (re-)imbibition, respectively. Respiration was
measured as the State 3 rate using NADH (A and B) or succinate (C and D) as substrate and as AOX capacity using NADH as substrate after activation
by pyruvate and DTT (E and F). The data are means ± standard error of mean (n = 3) and lines are regressed with Eqn 2.
in the dry state. During germination, seed must initiate
the process of recovery for mitochondrial intactness
and function (Bewley 1997). This recovery requires
the active participation of many physiological and
biochemical processes, such as gene expression in both
Physiol. Plant. 144, 2012
nucleus and mitochondria, protein synthesis, import
and modification (Bewley 1997, Howell et al. 2006).
If one of these processes is interrupted by desiccation,
mitochondrial recovery may become impossible. Thus
monitoring the recovery of mitochondrial functions
29
A
B
Fig. 7. The relationships between survival percentage of seeds and
recovery of structure and function of embryo axis mitochondria during
imbibition and desiccation of seeds. The seed survival data include the
survival of untreated seeds (CK), seeds imbibed in distilled water for 12,
25 and 36 h and dehydrated (H12D, H25D and H36D) and seeds imbibed
in CaCl2 and MV for 25 h and dehydrated (Ca25D and MV25D). The
maximum State 3 (St3) respiration with NADH or succinate as substrate
(A), AOX respiration (A), IMM integrity (B) to be recovered in the CK and
imbibed–dehydrated seeds were estimated from the regressions in Figs
6 and 7, respectively. Linear models were applied to the relationships.
The seed survival related to the NADH-dependent St3 respiration
with y = 229.1 + 3.854x (A, solid line; R2 = 0.80); to the succinatedependent St3 respiration with y = 146.6 + 3.245x (A, dashed line;
R2 = 0.91); to the AOX capacity with two lines: y = 11.2 + 1.754x
(when x ≤ 50.20) and y = 151 + 1.038x (when x ≥ 50.20) (A, dotted
line; R2 = 0.74); to the uncoupled rate ratio [FCCP/State 4 (St4)] with
y = 2.553 + 0.0323x (B, solid line R2 = 0.93) and to the RCR (St3/St4)
with y = 1.578 + 0.0231x (B, dashed line; R2 = 0.80).
after desiccation can be a sensitive way of assessing
desiccation stress. Our results support this hypothesis.
We have here chosen to study the biochemical
properties of isolated and purified mitochondria. In this
way, we can monitor changes in membrane structure
and enzyme biosynthesis, the former by permeability
assays, the latter as changes in the proportion of
a given enzyme measured as activity per milligram
mitochondrial protein. This cannot be done in the
homogenate or in the crude mitochondria, because
an unknown proportion of these fractions is not
mitochondria. The question is to what extent the purified
30
mitochondria are representative of all the mitochondria
present in the tissue before extraction? We observed a
very good correlation between the latency of the purified
mitochondria and the crude mitochondria (Fig. S1B).
Yield calculated as total CCO activity in the purified
mitochondria relative to the crude mitochondria also
increases linearly with CCO latency (Fig. S1A). We
conclude that the properties of purified mitochondria
reflected those of the crude mitochondria reasonably
well.
Although the importance of mitochondrial recovery
for seed germination is well known (Benamar et al.
2003, Bewley 1997, Howell et al. 2006, Logan et al.
2001), we still do not know how to quantify this process,
making it difficult to compare the effect of different
treatments. We here attempt to apply a germination
model (Bradford 2002, Wang et al. 2009) to quantify
the mitochondrial recovery across a series of imbibition
times, and clearly show the extent and the time of
recovery of mitochondrial intactness and function in
pea seed embryo axis during germination (Figs 4–6).
Among the mitochondrial recoveries, OMM integrity
recovered first (within 18 h, Fig. 4C), then CCO activity
(Fig. 4A), IMM integrity (Fig. 5A, C), State 3 rate (Fig. 6A,
C) and AOX capacity (Fig. 6E). These recoveries were
impaired after loss of seed desiccation tolerance during
imbibition. Seeds imbibed in distilled water for 12 h
were desiccation tolerant (Fig. 2), and mitochondria in
H12D seed embryo axis recovered as well or even
better than CK mitochondria (Figs 3, 4A, C, 5A, C and
6A, C, E). In contrast, seeds imbibed in distilled water
for 36 h lost their desiccation tolerance (Fig. 2), and
recoveries in H36D mitochondria were slower and less
complete than in CK mitochondria (Figs 3, 4A, C, 5A,
C and 6A, C, E). When seed desiccation tolerance was
increased by incubating the seeds in a CaCl2 solution
(Fig. 2D), the recovery of mitochondrial intactness
and respiratory function was improved correspondingly
(Figs 5B, D and 6B, D). The recovery of IMM integrity
was clearly impaired in MV25D seed embryo axis
(Fig. 5B, D), consistent with the observed decreased
desiccation tolerance of the seeds (Fig. 2D). These results
indicate that the recovery of mitochondrial intactness
and respiratory function is important for seed desiccation
tolerance.
The extent to which the mitochondrial intactness
and function, especially the IMM integrity and State 3
respiration, could be recovered determined the survival
of seeds (Fig. 7). It is perhaps not surprising that seed
survival depends on the intactness of the embryo
axis mitochondria measured as proton permeability
(Fig. 7B), because this parameter determines the ability
of the mitochondria to produce ATP. However, it
Physiol. Plant. 144, 2012
is interesting that seed survival is linearly correlated
with the respiratory rate of isolated embryo axis
mitochondria measured per milligram of mitochondrial
protein (Fig. 7A). This implies that the mitochondrial
recovery required for seed survival is affected by
insertion of electron transport proteins into pre-existing
mitochondria rather than by mitochondrial proliferation.
Cellular membranes are one of the major sites of
desiccation injury (Leprince et al. 1993). Benamar et al.
(2003) showed that mitochondrial membranes are the
primary targets for ageing injuries and possible also for
desiccation damage. However, they did not follow the
germination process or the recovery for longer than 22 h,
so it is not clear whether the incomplete recovery they
observed after ageing was indeed incomplete or whether
it was because of a delayed recovery. We monitored
mitochondrial membrane integrity during the whole
germination process, and found that IMM integrity and its
recovery (Fig. 5) were more sensitive to desiccation than
those of OMM (Fig. 4). OMM integrity reached 96% in
all treatments although the recovery was slowed down
in H36D mitochondria (Fig. 4C), while IMM integrity
in H25D and H36D mitochondria never recovered to
the same levels as in CK mitochondria even after 30 h
(re-)imbibition (Fig. 5).
Ca2+ is very important for the structure and function
of plant cells and plant membranes (White and Broadley
2003). It is also an important second messenger in
plant cell in response to many abiotic stresses (Kudla
et al. 2010, Lecourieux et al. 2006, Sanders et al. 2002).
There is little information about the effect of Ca2+
on seed desiccation stress. We found that imbibition
treatment with CaCl2 markedly improved the subsequent
desiccation tolerance of seeds (Fig. 2C) and the effect
increased with increasing CaCl2 concentrations (data not
shown). A positive effect of CaCl2 on seed desiccation
tolerance has also been found in soybean (Glycine
max ) seeds (Song et al. 2003). The observed increase in
desiccation tolerance by application of CaCl2 (Fig. 2C)
may result from the improved recovery of mitochondrial
CCO activity (Fig. 4B), IMM integrity (Fig. 5B, D) and
respiration (Fig. 6B, D).
Ca2+ has several roles in plant mitochondria. It
is bound to the mitochondrial membranes where it
probably contributes to stability and function (Møller
1983, Møller et al. 1981). Ca2+ is required for activity
by a number of enzymes including the external NADH
dehydrogenase in the respiratory chain in some species
(Møller 2001, Møller et al. 1981, Pical et al. 1993).
NADH oxidation by pea leaf mitochondria is Ca2+
dependent (Nash and Wiskich 1983), which we can
confirm for pea embryo axis mitochondria (see section
Results). Under the assay conditions used in this study,
Physiol. Plant. 144, 2012
there was sufficient Ca2+ bound to the membranes to
give maximum rates of NADH oxidation, similar to
what has been observed previously, e.g. on Jerusalem
artichoke mitochondria (Møller et al. 1981).
The improved recovery of CCO activity, IMM integrity
and respiration in Ca25D seeds (Figs 4B, D, 5B, D
and 6B, D, F) could be connected with the above
direct effects of Ca2+ on the mitochondria. The effect
could also be more indirect. Ca2+ can activate defense
gene expression via signal transduction (Kudla et al.
2010, Lecourieux et al. 2006, Sanders et al. 2002) and
CCO genes are induced during rice seed germination
in the presence of Ca2+ (Howell et al. 2006). Howell
et al (2007) indicated that oxygen deficit could decrease
protein import into mitochondria. Recently, Kuhn et al.
(2009) reported that Ca2+ is necessary for protein import
into the IMM (Kuhn et al. 2009). We speculate that
Ca2+ may also promote or maintain the protein import
pathway, and in that way improve mitochondrial repair.
Oxidative stress leads to ROS accumulation and
cellular damage (Bailly 2004, Møller et al. 2007). It
has often been proposed that the loss of desiccation
tolerance is because of ROS production (Finch-Savage
et al. 1994, Hendry et al. 1992, Li and Sun 1999,
Roach et al. 2010, Wu et al. 2009). In this study, we
imbibed the seeds in a solution containing MV, a
ROS producer (Halliwell and Gutteridge 2007), to test
whether increased oxidative stress would affect the seed
desiccation tolerance and the recovery of mitochondrial
structure and function. The MV treatment markedly
decreased the seed survival percentage (Fig. 2C) and
the ability to recover IMM coupling during re-imbibition
(Fig. 6B, D). MV treatment did not decrease the viability
of imbibed seeds, but increased the sensitivity of the
imbibed seeds to dehydration (Fig. 2D). We conclude
that MV treatment does make pea seeds less desiccation
tolerant and that this might be because of increased
mitochondrial ROS production.
AOX activity is induced and increased in response
to plant stress, such as cold and heat temperature
(Armstrong et al. 2008, Fiorani et al. 2005, GonzàlezMeler et al. 1999, Rachmilevitch et al. 2007, Watanabe
et al. 2008), drought (Bartoli et al. 2005, Ribas-Carbo
et al. 2005) and pathogen attack (Maxwell et al. 1999,
Simons et al. 1999). The capacity of the AOX pathway
was low in mitochondria from CK pea seed embryo
axes early in (re-)imbibition (germination) (Fig. 6E, F).
Imbibition followed by desiccation had only a relatively
small effect on AOX capacity except in H25D where the
effect was larger (Fig. 6E). In the presence of CaCl2 or
MV in the imbibition solution, AOX capacity was further
increased (Fig. 6F). As MV increases ROS production
in the tissue, this could be the cause of the increased
31
AOX induction. Further studies of the effects of MV on
mitochondrial ROS production and the consequences
for mitochondrial structure and function are required.
In most of the mitochondria, AOX capacity was only
10–30% of the State 3 rate. Exceptions were CK and
H36D early during (re-)imbibition where AOX was
60–100% of State 3 with NADH (and pyruvate) (Fig. S2).
This indicates that the AOX does not protect the mitochondria against desiccation damage, but rather against
damage during the early imbibition phase, when ROS is
produced (Bailly 2004). However, the effect of AOX is
not so strong that it can improve mitochondrial recovery.
Conclusions
The loss of desiccation tolerance during imbibition in
pea seeds is correlated with a decrease in IMM integrity
and cytochrome respiratory function during dehydration
and in the subsequent recovery of these functions
during re-imbibition. Ultimately, the mitochondria may
be unable to provide the cell with sufficient energy
and metabolic intermediates to maintain normal cellular
physiology. The positive influence of the CaCl2 treatment
on seed desiccation tolerance may well be due to
the requirement for Ca2+ in membrane structure and
function. In contrast, the negative effects of MV treatment
on recovery may be due to ROS-induced damage.
Acknowledgements – This work was supported by the
National Natural Sciences Foundation of China (30870223).
The authors are grateful to Jing-Hua Wu (Institute of
Botany, The Chinese Academy of Sciences) for helping
excise embryo axes from seeds. IMM was supported by
a Chinese Academy of Sciences Visiting Professorship for
senior international scientists.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Correlation of latency of crude mitochondrial
CCO activity with (A) yield of purified mitochondria
(PM) and (B) latency of PM CCO activity.
Fig. S2. Percentage of AOX to State 3 respiration in pea
embryo axis mitochondria during (re-)imbibition.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
Edited by P. Gardeström
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Physiol. Plant. 144, 2012