the role of recovery of mitochondrial ... -...

Physiologia Plantarum 144: 20–34. 2012 Copyright © Physiologia Plantarum 2011, ISSN 0031-9317

The role of recovery of mitochondrial structure and functionin desiccation tolerance of pea seedsWei-Qing Wanga, Hong-Yan Chenga, Ian M. Møllera,b and Song-Quan Songa,∗

aGroup of Seed Physiology and Biotechnology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, ChinabDepartment of Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark

Correspondence*Corresponding author,e-mail: [email protected]

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 theirdesiccation tolerance in parallel. To gain a better understanding of thisprocess, we studied the recovery of mitochondrial structure and function inpea (Pisum sativum cv. Jizhuang) seeds with different tolerance to desiccation.Mitochondria were isolated and purified from the embryo axes of controland imbibed–dehydrated pea seeds after (re-)imbibition for various times.Recovery of mitochondrial structure and function occurred both in controland imbibed–dehydrated seed embryo axes, but at different rates and todifferent maximum levels. The integrity of the outer mitochondrial membranereached 96% in all treatments. However, only the seeds imbibed for 12 hand then dehydrated recovered the integrity of the inner mitochondrialmembrane (IMM) and State 3 (respiratory state in which substrate and ADPare present) respiration (with NADH and succinate as substrate) to the controllevel after re-imbibition. With increasing imbibition time, the degree towhich each parameter recovered decreased in parallel with the decreasein desiccation tolerance. The tolerance of imbibed seeds to desiccationincreased and decreased when imbibed in CaCl2 and methylviologensolution, respectively, and the recovery of the IMM integrity similarlyimproved and weakened in these two treatments, respectively. Survival ofseeds after imbibition–dehydration linearly increased with the increase inability to recover the integrity of IMM and State 3 respiration, which indicatesthat recovery of mitochondrial structure and function during germination hasan important role in seed desiccation tolerance.

Introduction

Desiccation tolerance generally refers to the ability oforganisms to survive loss of 80 to 90% of protoplasmicwater to reach <0.3 g water g−1 dry matter. Under theseconditions, there is no free water in the living cellsand the structure of macromolecules and membranes isprotected by specific mechanisms (Hoekstra et al. 2001,

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)propanesulphonicacid; 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 arepresent; State 4, respiratory state in which all ADP has been converted into ATP; TX, Triton X-100.

Oliver et al. 2000). This phenomenon is widespreadacross the plant kingdom, including ferns, mosses,pollen, angiosperm resurrection plants and orthodoxseeds. In orthodox seeds, desiccation tolerance isacquired during seed maturation approximately halfwaythrough development. This trait ensures that seeds passunharmed through maturation drying and retain viabilityin the dry state for long periods of time under natural

20 Physiol. Plant. 144, 2012

conditions (Kermode and Finch-Savage 2002, Vertucciand Farrant 1995).

Bewley (1979) suggested that three protoplasmic prop-erties are required for plant or plant tissue to establishdesiccation tolerance: (1) minimize damage from desic-cation and/or rehydration, (2) maintain cellular integrityin the dry state and (3) mobilize repair mechanismson rehydration. These criteria highlight the importanceof two basic cellular process, protection and repair,in desiccation tolerance (Oliver et al. 2005). Manyspecific protective mechanisms have been implicatedin acquisition and maintenance of seed desiccationtolerance, including reduction of the degree of vac-uolization, intracellular de-differentiation, ‘switching off’metabolism, accumulation of protective molecules, suchas late embryogenesis abundant (LEA) proteins, sucroseand certain oligosaccharides, as well as the presenceand efficient operation of antioxidant systems (Berjakand Pammenter 2008, Pammenter and Berjak 1999).Although repair mechanisms have been identified inmosses 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 organellefor energy supply and its function is tightly coupledto seed germination (Bewley 1997). During matura-tion drying, seed mitochondria disassemble because ofdesiccation stress and in electron micrographs of dryseeds the mitochondria appear with poorly differenti-ated structure (Ehrenshaft and Brambl 1990, Logan et al.2001, Morohashi et al. 1981). A process of repair ofmitochondrial structure and function is necessary forseed germination during the imbibition process (Bewley1997). Many studies have shown that mitochondria indry seeds recover the mature structure with well-definedcristae and a dense matrix during the germination pro-cess (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 theenergy and metabolic intermediates that seed germina-tion requires.

Mitochondria are one of the targets for various stressdamage, probably because of their large turnover ofreactive 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 thatthe ability of maize seedlings to recover from chillinginjury was, at least in part, because of the recovery ofmitochondrial function. Severe seawater stress decreasesdurum wheat germination and delays seedling growth,and damages the mitochondrial succinate-dependentoxidative phosphorylation at the same time (Flagellaet al. 2006). It is possible that loss of desiccation

tolerance in seeds is because of desiccation damageto their mitochondria. Indeed, the respiration rate andactivity of cytochrome c oxidase (CCO) and malatedehydrogenase in mitochondria from axes of recalcitrantAntiaris toxicaria seeds and from embryos of orthodoxmaize seeds all decreased during dehydration (Songet al. 2009, Wu et al. 2009), but we do not have anyinformation about their recovery during germination. Inview of the importance of mitochondrial repair for seedgermination, we here hypothesize that this process isalso necessary for seed desiccation tolerance.

When mature seeds are imbibed and then dehydrated,survival decreases gradually with increasing imbibitiontime, that is, desiccation tolerance is lost duringseed germination (Dasgupta et al. 1982, Koster andLeopold 1988, Reisdorph and Koster 1999, Senaratnaand McKersie 1983). Thus germinating orthodox seedsresemble recalcitrant seeds, and this system has thereforebeen suggested to be a useful model for understandingthe mechanisms of recalcitrance (Sun 1999). In nature,seeds may experience several imbibition–desiccationcycles during germination because of fluctuations insoil moisture. After the point at which the seedshave lost their desiccation tolerance, these cycles willdecrease seed germination and seedling formation. Thusunderstanding desiccation tolerance will be importantfor optimizing not only storage of recalcitrant seeds, butalso crop yield.

Desiccation tolerance can be improved in germinatingseeds by treatment with certain exogenous chemicals,such as polyethylene glycol or abscisic acid (Brugginkand Vandertoorn 1995, Buitink et al. 2003). This methodcan be very useful for studying the mechanism ofdesiccation tolerance (Faria 2006). Production of ROSduring dehydration is considered as one of the reasonscausing desiccation damage (Bailly 2004). Thus theapplication of a ROS producer, such as methylviologen(MV) to desiccation-tolerant seeds would be expectedto decrease or remove the tolerance. Ca2+ acting asa second messenger can improve the response ofplant to many adverse stresses (Kudla et al. 2010,Lecourieux et al. 2006, Sanders et al. 2002). Ca2+ isalso important for membrane structure (Møller 1983,White and Broadley 2003), but little is known about itseffect on desiccation tolerance.

In this study, we have investigated the change of des-iccation 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 toler-ance of germinating seeds. Mitochondria were isolatedand purified from the embryo axes of the untreatedseeds and of a series of imbibed–dehydrated seeds

Physiol. Plant. 144, 2012 21

(tolerant, partly tolerant and intolerant to desiccation)and used to monitor the recovery of mitochondrial struc-ture and function during seed (re-)imbibition. A normalcumulative equation was applied to fit the time coursesof mitochondrial parameters during (re-)imbibition. Ourresults indicated that the recovery of mitochondrial struc-ture and function during germination is important in seeddesiccation tolerance.

Materials and methods

Plant material

Pea (P. sativum cv. Jizhuang) seeds were obtained fromGuangling Seed Company (Datong, Shanxi, China) inSeptember 2008 and were kept in plastic bags at 5◦Cuntil used. The water content of the seeds was about0.10 g H2O g−1 dry weight.

Seed imbibition, dehydration and survival

Seed imbibition and germination

Three replicates of 25 pea seeds each were placed ontwo layers of filter paper and imbibed in 40 ml distilledwater or treatment solution (20 mM CaCl2 or 2 mMMV both dissolved in distilled water) in closed 15-cmdiameter Petri dishes at 20◦C in darkness for up to 48 h.Radicle protrusion of 2 mm was used as the criterion ofgermination, and the number of germinated seeds wascounted regularly throughout the test period.

Another sample of three replicates of 25 pea seedseach was imbibed in distilled water or treatment solu-tion (20 mM CaCl2 or 2 mM MV) to determine seedwater uptake. These seeds were regularly removed fromthe Petri dishes to determine the seed weight, which wasused to calculate the seed water content.

Dehydration and survival

The pea seeds were imbibed in distilled water ortreatment solution (20 mM CaCl2 or 2 mM MV) at 20◦Cin darkness for 12–48 h, and then dehydrated to a watercontent of about 0.10 g g−1 by burying them in activatedsilica gel [1:8 (v:v) seeds:silica gel] within a closed plasticbox for 24–36 h.

The untreated (control) and imbibed–dehydratedseeds were germinated on two layers of filter papermoistened with 40 ml of distilled water in Petri dishes(15-cm diameter) at 20◦C in darkness for 7 days tomeasure seed survival. Likewise, seeds imbibed in CaCl2or MV for 25 h were tested for germination and survival.Seeds showing a normal epicotyl and primary radicalwere considered as being alive.

The control seeds (CK) imbibed in distilled water for4, 12, 18, 24, 30 and 36 h, and the seeds imbibed indistilled water for 12, 25 or 36 h and dehydrated (H12D,H25D or H36D), as well as seeds imbibed in CaCl2 orMV for 25 h and dehydrated (Ca25D or MV25D) werere-imbibed in distilled water for 4, 8, 12, 18, 24 and30 h and then used for the isolation and purification ofmitochondria (Fig. 1)

Determination of water content

The water content of seeds was determined accordingto the method of International Seed Testing Association(1999) and expressed on a dry mass basis (g H2O g−1

dry weight).

Transmission electron microscopy

Embryo axes of CK, H12D and H36D (labels seeFig. 1) seeds were used for fixation directly or after(re-)imbibition of the seeds for 24 h, respectively.

Excision of embryo axis and purification of mitochondria

Imbibition for4, 12, 18, 24, 30, 36 h

Re-imbibition for4, 8, 12, 18, 24, 30 h

Dehydration(H12D)

Dehydration(H25D Ca25D MV25D)

Dehydration(H36D)

No dehydration(CK)

Imbibition for 12 h Imbibition for 25 h in Imbibition for 36 hNo imbibition

Control seeds (CK)

distilled water CaCl2 MV

Fig. 1. Protocol used for the imbibition, dehydration and subsequent re-imbibition of pea seeds. CK, H12D, H25D, Ca25D, MV25D and H36D aredry seeds with a water content of about 0.10 g g−1.


Mitochondria isolated and purified from embryo axesof the seeds (re-)imbibed for 24 h were also used. Theembryo axes and purified mitochondria were fixed in2.5% (v/v) glutaraldehyde, 100 mM sodium phosphate(pH 7.2) (for purified mitochondria, the solution also con-tained 0.4 M sucrose) for 2 h. The fixed embryo axes thathad been rinsed (twice) and purified mitochondria thathad 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, dehydratedin an ethanol series and then embedded in Epon resin.Ultra-thin sections were cut and stained with uranylacetate followed by lead citrate before observation. Theelectron micrographs were recorded with a JEM-1230transmission electron microscope (JEOL, Tokyo, Japan).

Isolation and purification of mitochondria

Mitochondria were prepared according to the method ofBenamar et al. (2003) modified as follows: 200 embryoaxes were homogenized using a precooled mortar andpestle in 20 ml of extraction buffer composed of 0.4 Msucrose, 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 withKOH. After washing the mortar with 20 ml extractionbuffer, the total 40 ml homogenate was filtered throughfour layers of gauze. The filtrate was centrifuged at 100 gfor 5 min and 500 g for 10 min, and the supernatant wasthen centrifuged at 15 000 g for 20 min. The resultingpellet was suspended in washing buffer composed of0.4 M mannitol, 1 mM EDTA, 0.2% (w/v) BSA, 10 mMMOPS–KOH, pH 7.2 and layered on to a discontinuousPercoll gradient: 3 ml of 25% Percoll containing 0.4 Mmannitol, 5 mM MgCl2, 0.2% BSA, 10 mM MOPS-KOH,pH 7.2, on top of 3 ml of 35% Percoll containing 0.4 Msucrose, 5 mM MgCl2, 0.2% BSA, 10 mM MOPS-KOH,pH 7.2. After centrifugation at 10 000 g for 20 min, themitochondrial band at the interface between 25 and 35%Percoll solution was collected and diluted eight timeswith washing buffer (without BSA), and then pelleted at13 000 g for 15 min to remove the Percoll. This dilutionand pelletation was repeated once. The pellets werefinally resuspended in washing buffer (without BSA) andused for assay of enzyme activity and determination ofoxygen consumption and protein content. All procedureswere carried out at 4◦C.

Assay of CCO activity

Activity of CCO (EC 1.11.1.5) was assayed using themethod of Møller et al. (1987), which measures the

oxidation of reduced cytochrome c at 550 nm. Thelatency of CCO activity was calculated as described byMøller et al. (1987).

Latency (%) = 100 × [(rate + TX) − (rate − TX)]/(rate + TX), (1)

where (rate + TX) and (rate − TX) are the oxidationrate of reduced cytochrome c by CCO in the reactionmedium with and without 0.05% (w/v) Triton X-100(TX), respectively.

Measurements of oxygen consumption

Oxygen consumption of mitochondria was measuredusing an oxygen electrode system (Oxytherm, HansatechInstruments, Pentney, King’s Lynn, Norfolk, UK). Thereaction medium contained 0.4 M mannitol, 10 mMKH2PO4, 10 mM KCl, 5 mM MgCl2 and 0.1% (w/v)BSA, 10 mM MOPS-KOH, pH 7.5, in a total volumeof 1 ml. For substrate oxidation, first mitochondria(80–150 μg protein) and the substrate (1.5 mM NADHor 10 mM succinate) was added followed by twoadditions of ADP, the first time (50 μM) to activateand the second time (100 μM) to obtain States 3and 4 respiration and to give the ADP/O ratio. A1 mM EGTA was added before NADH in some controlexperiments to remove Ca2+ from the membranes,which might be sufficient to give maximal NADHoxidation activity (Møller 1983, Møller et al. 1981,Nash and Wiskich 1983). Uncoupler, carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP; 2 μM) wasused to uncouple NADH oxidation to obtain anothermeasure of uninhibited respiration rate. For estimationof alternative oxidase (AOX) capacity, the cytochromeoxidase pathway during NADH oxidation was inhibitedby potassium cyanide (1 mM), pyruvate (10 mM) anddithiothreitol (DTT; 10 mM) were added to activateAOX, and finally salicylhydroxamic acid (SHAM; 2 mM)

was added to inhibit AOX. AOX capacity was calculatedas the rate after addition of DTT and pyruvate minusthe rate after SHAM addition (= residual respiration).The mitochondrial respiration was expressed as nmolO2 mg protein−1 min−1.

Determination of protein

The concentration of protein in the purified mitochondriawas determined according to Bradford (1976), using BSAas a standard.

Statistical analysis

A modified population based model (Eqn 2) was appliedto fit the time courses of recovery of mitochondrial


A

C D

B

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 (H2O),CaCl2 and MV. (B) Germination of seeds imbibed for 0–48 h in H2O, CaCl2 and MV. (C) Survival of seeds after imbibition and dehydration. Seedsimbibed in H2O, 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 ofmoist filter paper at 20◦C for 10 days to detect survival. (D) The survival of seeds before and after dehydration. Seeds imbibed in H2O, MV or CaCl2for 25 h were directly germinated in distilled water (solid bar) or were dehydrated first and then germinated in distilled water (open bar) for 10 daysto 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 themodel describing seed germination and dormancyrelease (Bradford 2002, Wang et al. 2009), assumes thatrecovery of the mitochondrial structure and functionduring (re-)imbibition follows a cumulative normaldistribution:

y = ymax × �[(x − μ)/σ ] (2)

where y is a parameter reflecting mitochondrial structureand function at a given imbibition or re-imbibition timex, ymax is the maximum value of y, � is the normalprobability integral and μ and σ are mean and standarddeviation of original distribution of y.

GRAPHPAD PRISM 5.0 (GRAPHPAD Software, San Diego,CA) was applied for nonlinear regression. F test withPRISM 5.0 was used to test whether data sets of differenttreatments could be shared with a global curve (Motulskyand Christopoulos 2003).

Results

Water uptake, germination and desiccationtolerance during imbibition

Water uptake of pea seeds showed a triphasic behaviorwith a rapid initial water uptake at the first 10 h, followedby a slower uptake between 10 and 30 h and finally aslightly faster phase after 30 h (Fig. 2A). Water uptakein distilled water, Ca2+ solution and MV solution wasthe same for the first 15 h and at 25 h, when seeds weredried, 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 whenimbibed in distilled water (Fig. 2B). Germination wasdelayed in the presence of CaCl2 (important forstructure) and MV (causes ROS formation), and the finalgermination percentage was also reduced by 10% in thepresence of MV in the imbibition medium (Fig. 2B).


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 frompea 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) werenot (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 axeswere excised and fixed. A, B, D, E, G and H show transmission electron micrographs of mitochondria in embryo axes cell, and the insert figures aretransmission electron micrographs of embryo axis cells at lower magnification. The embryo axes of seeds re-imbibed for 24 h were also excised andused 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 μmand 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 hof imbibition, after which seed death occurred ina log cumulative normal distribution over imbibitiontime (Fig. 2C). No seeds survived after imbibition for48 h followed by dehydration (Fig. 2C). At the sameimbibition time followed by dehydration, the presenceof CaCl2 in the imbibition solution increased, and MVdecreased, the survival percentage of seeds, as indicatedby the shift in the T50 value (Fig. 2C). Imbibition inCaCl2 and MV for 25 h did not in itself affect seedviability, as seeds imbibed in these two solutions andthen germinated without an intervening dehydration allsurvived (Fig. 2D).

Transmission electron micrographsof mitochondria

A morphological examination of mitochondria in situand after isolation was performed using transmissionelectron microscopy (Fig. 3). In the control seeds (CK,Fig. 3A) and imbibed–dehydrated seeds (H12D andH36D, Fig. 3D, G), mitochondria had little discernibleinternal structure. After 24 h of re-imbibition, themitochondrial structure was repaired, but only CK andH12D had fully differentiated mitochondria (Fig. 3B,C, E, F) and in H36D seeds the mitochondria couldnot recover the structure with well-defined cristae(Fig. 3H, I). The embryo axis cells in H36D seedsbecame plasmolyzed after dehydration (Fig. 3G insert),


and the plasmolyzed cells were still plasmolyzed aftersubsequent rehydration (Fig. 3H insert). This occurreddespite the fact that the embryo axes were fully imbibedas seen from the high water content of the H36D axes(Fig. 3H insert) in comparison with CK and H12D axesafter 24 h imbibition (Fig. 3B, E insert). Thus there maybe desiccation damage to the plasma membrane in theH36D seeds.

Purification of embryo axis mitochondria

Mitochondria were isolated and purified from embryoaxes of germinating pea seeds [0–36 h (re-)imbibition]pretreated in six different ways: control seeds (CK), seedsimbibed in distilled water for 12, 25 or 36 h and thendehydrated (H12D, H25D or H36D), or seeds imbibedin CaCl2 or MV for 25 h and then dehydrated (Ca25D orMV25D) (Fig. 1).

Mitochondrial purity was monitored in crude andpurified mitochondria by the specific activity of CCO,an enzyme found only in the inner mitochondrialmembrane (IMM; Lardy and Ferguson 1969). The latencyof CCO activity was used to monitor the integrity of theouter mitochondrial membrane (OMM), as an intactouter membrane does not permit the CCO substrate,reduced cytochrome c (12.5 kDa), to be oxidized byCCO (Neuburger 1985).

The CCO specific activity increased 2.2 to 6.5-fold from crude mitochondria to purified mitochondria(results not shown). This was mainly because of alarge variation in the percentage of total CCO activityin the crude mitochondria recovered in the purifiedmitochondria. The recovery was linearly correlated withOMM integrity (Fig. S1A, supporting information). OMMintegrity in purified mitochondria was linearly correlatedwith that of crude mitochondria (Fig. S1B) and it did notchange as a result of purification (see x = y line inFig. S1B). In summary, when the OMM of the crudemitochondria was relatively intact, a large percentage ofthe 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 fromCK seeds at 6 and 12 h of imbibition, but increasedrapidly thereafter (Fig. 4A). CCO activity was higherin mitochondria from the imbibed–dehydrated seedsH12D, H25D and H36D than in mitochondria fromCK seeds for the first 18 h of (re-)imbibition (Fig. 4A).However, the maximum CCO activity to be recoveredwas similar in CK, H12D and H25D seeds, andsignificantly lower in H36D seeds (Fig. 4A). Compared

to H25D seeds, CaCl2 treatment (Ca25D) increased CCOactivity at all times of re-imbibition, while MV treatment(MV25D) had no effect (Fig. 4B).

In CK mitochondria, CCO latency increased veryrapidly and reached 96% within 18 h of imbibition. Inimbibed–dehydrated seeds, the final CCO latency wassimilar to that in CK seeds (Fig. 4C, D), whereas it tooklonger to recover in H36D seeds (Fig. 4C). Pretreatmentof the seeds with CaCl2 or with MV (followed bydehydration) had no effect on the recovery of CCOlatency during germination (Fig. 4D).

IMM integrity

The functional intactness of the IMM was estimatedby measuring the State 3 (respiratory state in whichsubstrate and ADP are present)/State 4 (respiratory statein which all ADP has been converted into ATP) ratio,the respiratory control ratio (RCR), as this ratio will berelatively high when proton leakage across the IMMis low. A similar parameter is uncoupled rate ratio(FCCP/State 4).

Among seeds imbibed in H2O, the recovery of IMMintegrity as indicated by RCR and the uncoupled rateratio, was more rapid in embryo axis mitochondriafrom the H12D and H25D seeds than in the CKmitochondria. However, only mitochondria from H12Dseeds could recover IMM integrity to the same highlevel as the CK mitochondria (Fig. 5A, C). The degree ofIMM integrity after 36 h re-imbibition was lower forH25D mitochondria and markedly lower for H36Dmitochondria (Fig. 5A, C). CaCl2 treatment (Ca25D)promoted, while MV treatment (MV25D) weakened,the recovery of the IMM integrity during re-imbibitioncompared to H2O treatment (H25D; Fig. 5B, D).

Respiration

A high respiratory activity per milligram mitochondrialprotein (relative to that of mitochondria purified frompea leaves – Nash and Wiskich 1983) indicates thatthe mitochondria are well able to contribute to seedmetabolism.

The addition of Ca2+ to mitochondria oxidizingNADH in the standard medium did not change therate of oxygen consumption measured. However, inthe presence of EGTA the rate was reduced by 85%indicating that external NADH oxidation in peed seedmitochondria is dependent on Ca2+ (results not shown).The NADH oxidation rates presented in Fig. 6A, B arethe maximum rates measured in the absence of EGTA.

State 3 respiration in CK mitochondria (NADH orsuccinate as substrate) was very low at 4 h re-imbibition,but increased rapidly to reach very high activities


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 andpurification of mitochondria. CCO activity of purified mitochondria was assayed (A and B) and the latency of the CCO activity (C and D) was calculatedto 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 thelatency 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 H36Dmitochondria, State 3 respiration was higher than therespiration in control mitochondria at the start time, butonly H12D mitochondria reached the same maximumlevel as the CK mitochondria after 30 h re-imbibition.For H25D and H36D mitochondria, the rates after 30 hwere only 73 and 48% of CK mitochondria (NADHas substrate; Fig. 6A) or 81 and 40% (succinate assubstrate; Fig. 6C) of CK mitochondria. State 3 respirationrecovered better in mitochondria from Ca25D seedembryo axes than in mitochondria from H25D embryoaxes (Fig. 6B, D). Although the State 3 respiration ofMV25D mitochondria was a little higher than for H25Dmitochondria early during re-imbibition, the maximumrate of respiration that the mitochondria could reachwas similar (Fig. 6B, D). Recovery of AOX capacity

was similar among CK, H12D and H36D mitochondria(Fig. 6E), but more rapid and reaching higher activities inH25D mitochondria (Fig. 6E). AOX capacity was higherin Ca25D and MV25D mitochondria than in controlmitochondria (Fig. 6F). Generally, AOX capacity wasonly a small part of the State 3 activity (Fig. S2), butin CK mitochondria after 4 h re-imbibition they werealmost the same size (Fig. S2A).

We calculated the ADP/O ratio during NADHoxidation and the values were all between 1.0 and1.3 with no clear trend (results not shown).

Seed survival and mitochondrial recovery

According to the Eqn 2, the maximum integrity ofIMM and oxygen consumption to be recovered during


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 andpurified 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 areregressed with Eqn 2.

(re-)imbibition was calculated and plotted against thepercentage of seed survival (Fig. 7). Seed survivalincreased linearly with increase in the NADH- orsuccinate-dependent State 3 respiration, uncoupled rateratio and RCR (Fig. 7A, B), whereas the pattern with AOXcapacity was not clear (Fig. 7A).

Discussion

Orthodox seeds acquire desiccation tolerance duringmaturation and lose it during imbibition (Berjak andPammenter 2008, Kermode and Finch-Savage 2002). Inthis, pea (P. sativum cv. Jizhuang) seeds gradually losttheir desiccation tolerance after imbibition for longerthan 15 h, and no seed survived (measured as theability to germinate within 7 days) after imbibition for

48 h followed by desiccation (Fig. 2C). The desiccationtolerance of imbibed seeds was improved and impairedwhen imbibition took place in CaCl2 and MV solutions,respectively, instead of in distilled water (Fig. 2C).When seeds were imbibed with CaCl2, a small delayin germination was found in comparison with seedsimbibed in water. This may be because of the slight saltstress caused by the CaCl2 solution.

One of the key functions of mitochondria is tocatalyze substrate oxidation and produce ATP for usein cellular metabolism. Mitochondria are affected byenvironmental 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


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 fromembryo axes of CK, H12D, H25D and H36D (A and C), and of CaD25 and MVD25 (B and D) seeds after (re-)imbibition, respectively. Respiration wasmeasured 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 activationby 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 initiatethe process of recovery for mitochondrial intactnessand function (Bewley 1997). This recovery requiresthe active participation of many physiological andbiochemical processes, such as gene expression in both

nucleus and mitochondria, protein synthesis, importand modification (Bewley 1997, Howell et al. 2006).If one of these processes is interrupted by desiccation,mitochondrial recovery may become impossible. Thusmonitoring the recovery of mitochondrial functions


A

B

Fig. 7. The relationships between survival percentage of seeds andrecovery of structure and function of embryo axis mitochondria duringimbibition and desiccation of seeds. The seed survival data include thesurvival of untreated seeds (CK), seeds imbibed in distilled water for 12,25 and 36 h and dehydrated (H12D, H25D and H36D) and seeds imbibedin CaCl2 and MV for 25 h and dehydrated (Ca25D and MV25D). Themaximum State 3 (St3) respiration with NADH or succinate as substrate(A), AOX respiration (A), IMM integrity (B) to be recovered in the CK andimbibed–dehydrated seeds were estimated from the regressions in Figs6 and 7, respectively. Linear models were applied to the relationships.The seed survival related to the NADH-dependent St3 respirationwith y = 229.1 + 3.854x (A, solid line; R2 = 0.80); to the succinate-dependent 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, dottedline; R2 = 0.74); to the uncoupled rate ratio [FCCP/State 4 (St4)] withy = 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 assessingdesiccation stress. Our results support this hypothesis.

We have here chosen to study the biochemicalproperties of isolated and purified mitochondria. In thisway, we can monitor changes in membrane structureand enzyme biosynthesis, the former by permeabilityassays, the latter as changes in the proportion ofa given enzyme measured as activity per milligrammitochondrial protein. This cannot be done in thehomogenate or in the crude mitochondria, becausean unknown proportion of these fractions is notmitochondria. The question is to what extent the purified

mitochondria are representative of all the mitochondriapresent in the tissue before extraction? We observed avery good correlation between the latency of the purifiedmitochondria and the crude mitochondria (Fig. S1B).Yield calculated as total CCO activity in the purifiedmitochondria relative to the crude mitochondria alsoincreases linearly with CCO latency (Fig. S1A). Weconclude that the properties of purified mitochondriareflected those of the crude mitochondria reasonablywell.

Although the importance of mitochondrial recoveryfor 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 differenttreatments. We here attempt to apply a germinationmodel (Bradford 2002, Wang et al. 2009) to quantifythe mitochondrial recovery across a series of imbibitiontimes, and clearly show the extent and the time ofrecovery of mitochondrial intactness and function inpea seed embryo axis during germination (Figs 4–6).Among the mitochondrial recoveries, OMM integrityrecovered 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 wereimpaired after loss of seed desiccation tolerance duringimbibition. Seeds imbibed in distilled water for 12 hwere desiccation tolerant (Fig. 2), and mitochondria inH12D seed embryo axis recovered as well or evenbetter than CK mitochondria (Figs 3, 4A, C, 5A, C and6A, C, E). In contrast, seeds imbibed in distilled waterfor 36 h lost their desiccation tolerance (Fig. 2), andrecoveries in H36D mitochondria were slower and lesscomplete than in CK mitochondria (Figs 3, 4A, C, 5A,C and 6A, C, E). When seed desiccation tolerance wasincreased by incubating the seeds in a CaCl2 solution(Fig. 2D), the recovery of mitochondrial intactnessand respiratory function was improved correspondingly(Figs 5B, D and 6B, D). The recovery of IMM integritywas clearly impaired in MV25D seed embryo axis(Fig. 5B, D), consistent with the observed decreaseddesiccation tolerance of the seeds (Fig. 2D). These resultsindicate that the recovery of mitochondrial intactnessand respiratory function is important for seed desiccationtolerance.

The extent to which the mitochondrial intactnessand function, especially the IMM integrity and State 3respiration, could be recovered determined the survivalof seeds (Fig. 7). It is perhaps not surprising that seedsurvival depends on the intactness of the embryoaxis mitochondria measured as proton permeability(Fig. 7B), because this parameter determines the abilityof the mitochondria to produce ATP. However, it


is interesting that seed survival is linearly correlatedwith the respiratory rate of isolated embryo axismitochondria measured per milligram of mitochondrialprotein (Fig. 7A). This implies that the mitochondrialrecovery required for seed survival is affected byinsertion of electron transport proteins into pre-existingmitochondria rather than by mitochondrial proliferation.

Cellular membranes are one of the major sites ofdesiccation injury (Leprince et al. 1993). Benamar et al.(2003) showed that mitochondrial membranes are theprimary targets for ageing injuries and possible also fordesiccation damage. However, they did not follow thegermination process or the recovery for longer than 22 h,so it is not clear whether the incomplete recovery theyobserved after ageing was indeed incomplete or whetherit was because of a delayed recovery. We monitoredmitochondrial membrane integrity during the wholegermination process, and found that IMM integrity and itsrecovery (Fig. 5) were more sensitive to desiccation thanthose of OMM (Fig. 4). OMM integrity reached 96% inall treatments although the recovery was slowed downin H36D mitochondria (Fig. 4C), while IMM integrityin H25D and H36D mitochondria never recovered tothe same levels as in CK mitochondria even after 30 h(re-)imbibition (Fig. 5).

Ca2+ is very important for the structure and functionof plant cells and plant membranes (White and Broadley2003). It is also an important second messenger inplant cell in response to many abiotic stresses (Kudlaet 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 imbibitiontreatment with CaCl2 markedly improved the subsequentdesiccation tolerance of seeds (Fig. 2C) and the effectincreased with increasing CaCl2 concentrations (data notshown). A positive effect of CaCl2 on seed desiccationtolerance has also been found in soybean (Glycinemax) seeds (Song et al. 2003). The observed increase indesiccation tolerance by application of CaCl2 (Fig. 2C)may result from the improved recovery of mitochondrialCCO activity (Fig. 4B), IMM integrity (Fig. 5B, D) andrespiration (Fig. 6B, D).

Ca2+ has several roles in plant mitochondria. Itis bound to the mitochondrial membranes where itprobably contributes to stability and function (Møller1983, Møller et al. 1981). Ca2+ is required for activityby a number of enzymes including the external NADHdehydrogenase 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 canconfirm for pea embryo axis mitochondria (see sectionResults). Under the assay conditions used in this study,

there was sufficient Ca2+ bound to the membranes togive maximum rates of NADH oxidation, similar towhat has been observed previously, e.g. on Jerusalemartichoke mitochondria (Møller et al. 1981).

The improved recovery of CCO activity, IMM integrityand respiration in Ca25D seeds (Figs 4B, D, 5B, Dand 6B, D, F) could be connected with the abovedirect effects of Ca2+ on the mitochondria. The effectcould also be more indirect. Ca2+ can activate defensegene expression via signal transduction (Kudla et al.2010, Lecourieux et al. 2006, Sanders et al. 2002) andCCO genes are induced during rice seed germinationin the presence of Ca2+ (Howell et al. 2006). Howellet al (2007) indicated that oxygen deficit could decreaseprotein import into mitochondria. Recently, Kuhn et al.(2009) reported that Ca2+ is necessary for protein importinto the IMM (Kuhn et al. 2009). We speculate thatCa2+ may also promote or maintain the protein importpathway, and in that way improve mitochondrial repair.

Oxidative stress leads to ROS accumulation andcellular damage (Bailly 2004, Møller et al. 2007). Ithas often been proposed that the loss of desiccationtolerance is because of ROS production (Finch-Savageet al. 1994, Hendry et al. 1992, Li and Sun 1999,Roach et al. 2010, Wu et al. 2009). In this study, weimbibed the seeds in a solution containing MV, aROS producer (Halliwell and Gutteridge 2007), to testwhether increased oxidative stress would affect the seeddesiccation tolerance and the recovery of mitochondrialstructure and function. The MV treatment markedlydecreased the seed survival percentage (Fig. 2C) andthe ability to recover IMM coupling during re-imbibition(Fig. 6B, D). MV treatment did not decrease the viabilityof imbibed seeds, but increased the sensitivity of theimbibed seeds to dehydration (Fig. 2D). We concludethat MV treatment does make pea seeds less desiccationtolerant and that this might be because of increasedmitochondrial ROS production.

AOX activity is induced and increased in responseto plant stress, such as cold and heat temperature(Armstrong et al. 2008, Fiorani et al. 2005, Gonzalez-Meler et al. 1999, Rachmilevitch et al. 2007, Watanabeet al. 2008), drought (Bartoli et al. 2005, Ribas-Carboet al. 2005) and pathogen attack (Maxwell et al. 1999,Simons et al. 1999). The capacity of the AOX pathwaywas low in mitochondria from CK pea seed embryoaxes early in (re-)imbibition (germination) (Fig. 6E, F).Imbibition followed by desiccation had only a relativelysmall effect on AOX capacity except in H25D where theeffect was larger (Fig. 6E). In the presence of CaCl2 orMV in the imbibition solution, AOX capacity was furtherincreased (Fig. 6F). As MV increases ROS productionin the tissue, this could be the cause of the increased


AOX induction. Further studies of the effects of MV onmitochondrial ROS production and the consequencesfor mitochondrial structure and function are required.

In most of the mitochondria, AOX capacity was only10–30% of the State 3 rate. Exceptions were CK andH36D early during (re-)imbibition where AOX was60–100% of State 3 with NADH (and pyruvate) (Fig. S2).This indicates that the AOX does not protect the mito-chondria against desiccation damage, but rather againstdamage during the early imbibition phase, when ROS isproduced (Bailly 2004). However, the effect of AOX isnot so strong that it can improve mitochondrial recovery.

Conclusions

The loss of desiccation tolerance during imbibition inpea seeds is correlated with a decrease in IMM integrityand cytochrome respiratory function during dehydrationand in the subsequent recovery of these functionsduring re-imbibition. Ultimately, the mitochondria maybe unable to provide the cell with sufficient energyand metabolic intermediates to maintain normal cellularphysiology. The positive influence of the CaCl2 treatmenton seed desiccation tolerance may well be due tothe requirement for Ca2+ in membrane structure andfunction. In contrast, the negative effects of MV treatmenton recovery may be due to ROS-induced damage.

Acknowledgements – This work was supported by theNational Natural Sciences Foundation of China (30870223).The authors are grateful to Jing-Hua Wu (Institute ofBotany, The Chinese Academy of Sciences) for helpingexcise embryo axes from seeds. IMM was supported bya Chinese Academy of Sciences Visiting Professorship forsenior international scientists.

References

Amirsadeghi S, Robson CA, Vanlerberghe GC (2007) Therole of the mitochondrion in plant responses to bioticstress. Physiol Plant 129: 253–266

Armstrong AF, Badger MR, Day DA, Barthet MM,Smith PMC, Millar AH, Whelan J, Atkin OK (2008)Dynamic changes in the mitochondrial electrontransport chain underpinning cold acclimation of leafrespiration. Plant Cell Environ 31: 1156–1169

Bailly C (2004) Active oxygen species and antioxidants inseed biology. Seed Sci Res 14: 93–107

Bartoli CG, Gomez F, Gergoff G, Guiamet JJ, Puntarulo S(2005) Up-regulation of the mitochondrial alternativeoxidase pathway enhances photosynthetic electrontransport under drought conditions. J Exp Bot 56:1269–1276

Benamar A, Tallon C, Macherel D (2003) Membraneintegrity and oxidative properties of mitochondriaisolated from imbibing pea seeds after priming oraccelerated ageing. Seed Sci Res 13: 35–45

Berjak P, Pammenter NW (2008) From Avicennia toZizania: seed recalcitrance in perspective. Ann Bot 101:213–228

Bewley JD (1979) Physiological aspects of desiccationtolerance. Annu Rev Plant Physiol Plant Mol Biol 30:195–238

Bewley JD (1997) Seed germination and dormancy. PlantCell 9: 1055–1066

Bradford MM (1976) Rapid and sensitive method forquantitation of microgram quantities of protein utilizingprinciple of protein-dye binding. Anal Biochem 72:248–254

Bradford KJ (2002) Applications of hydrothermal time toquantifying and modeling seed germination anddormancy. Weed Sci 50: 248–260

Bruggink T, Vandertoorn P (1995) Induction of desiccationtolerance in germinated seeds. Seed Sci Res 5: 1–4

Buitink J, Vu BL, Satour P, Leprince O (2003) There-establishment of desiccation tolerance in germinatedradicles of Medicago truncatula Gaertn. seeds. Seed SciRes 13: 273–286

Dasgupta J, Bewley JD, Yeung EC (1982) Desiccation-tolerant and desiccation-intolerant stages during thedevelopment and germination of Phaseolus vulgarisseeds. J Exp Bot 33: 1045–1057

Ehrenshaft M, Brambl R (1990) Respiration andmitochondrial biogenesis in germinating embryos ofmaize. Plant Physiol 93: 295–304

Faria JMR (2006) Desiccation tolerance and sensitivity inMedicago truncatula and Inga vera seeds. Thesis.University of Wageningen, Wageningen, TheNetherlands

Finch-Savage WE, Hendry GAF, Atherton NM (1994) Freeradical activity and loss of viability during drying ofdesiccation sensitive tree seeds. Proc Roy Soc Edinb SectB-Bio Sci 102: 257–260

Fiorani F, Umbach AL, Siedow JN (2005) The alternativeoxidase of plant mitochondria is involved in theacclimation of shoot growth at low temperature. A studyof Arabidopsis AOX1a transgenic plants. Plant Physiol139: 1795–1805

Flagella Z, Trono D, Pompa M, Di Fonzo N, Pastore D(2006) Seawater stress applied at germinationaffects mitochondrial function in durum wheat(Triticum durum) early seedlings. Funct Plant Biol 33:357–366

Gonzalez-Meler MA, Ribas-Carbo M, Giles L, Siedow JN(1999) The effect of growth and measurementtemperature on the activity of the alternative respiratorypathway. Plant Physiol 120: 765–772


Halliwell B, Gutteridge JMC (2007) Free Radicals inBiology and Medicine, 4th Edn. Oxford University Press,New York

Hendry GAF, Finch-Savage WE, Thorpe PC, Atherton NM,Buckland SM, Nilsson KA, Seel WE (1992) Free-radicalprocesses and loss of seed viability during desiccation inthe recalcitrant species Quercus robur L. New Phytol122: 273–279

Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms ofplant desiccation tolerance. Trends Plant Sci 6: 431–438

Howell K, Millar A, Whelan J (2006) Ordered assembly ofmitochondria during rice germination begins withpromitochondrial structures rich in components of theprotein import apparatus. Plant Mol Biol 60: 201–223

Howell KA, Cheng K, Murcha MW, Jenkin LE, Millar AH,Whelan J (2007) Oxygen initiation of respiration andmitochondrial biogenesis in rice. J Biol Chem 282:15619–15631

International Seed Testing Association (1999) Internationalrules for seed testing. Seed Sci Technol 27(suppl): 47–50

Kermode A, Finch-Savage WE (2002) Desiccationsensitivity in orthodox and recalcitrant seeds in relationto development. In: Black M, Pritchard H (eds)Desiccation and Survival in Plants: Drying withoutDying. CABI Publishing, Oxon, pp 237–271

Koster KL, Leopold AC (1988) Sugars and desiccationtolerance in seeds. Plant Physiol 88: 829–832

Kudla J, Batistic O, Hashimoto K (2010) Calcium signals:the lead currency of plant information processing. PlantCell 22: 541–563

Kuhn S, Bussemer J, Chigri F, Vothknecht UC (2009)Calcium depletion and calmodulin inhibition affect theimport of nuclear-encoded proteins into plantmitochondria. Plant J 58: 694–705

Lardy HA, Ferguson SM (1969) Oxidative phosphorylationin mitochondria. Annu Rev Biochem 38: 991–1034

Lecourieux D, Raneva R, Pugin A (2006) Calcium in plantdefence-signalling pathways. New Phytol 171: 249–269

Leprince O, Hendry GAF, McKersie BD (1993) Themechanisms of desiccation tolerance in developingseeds. Seed Sci Res 3: 231–246

Li CR, Sun WQ (1999) Desiccation sensitivity andactivities of free radical-scavenging enzymes inrecalcitrant Theobroma cacao seeds. Seed Sci Res 9:209–217

Logan DC, Millar AH, Sweetlove LJ, Hill SA, Leaver CJ(2001) Mitochondrial biogenesis during germination inmaize embryos. Plant Physiol 125: 662–672

Macherel D, Benamar A, Avelange-Macherel MH,Tolleter D (2007) Function and stress tolerance of seedmitochondria. Physiol Plant 129: 233–241

Maxwell DP, Wang Y, McIntosh L (1999) The alternativeoxidase lowers mitochondrial reactive oxygenproduction in plant cells. Proc Natl Acad Sci USA 96:8271–8276

Møller IM (1983) Monitoring of membrane-bound divalentcations in plant mitochondria using chlorotetracyclinefluorescence. Physiol Plant 59: 567–572

Møller IM (2001) Plant mitochondria and oxidative stress.Electron transport, NADPH turnover and metabolism ofreactive oxygen species. Annu Rev Plant Physiol PlantMol Biol 52: 561–591

Møller IM, Johnston SP, Palmer JM (1981) A specific rolefor Ca2+ in the oxidation of exogenous NADH byJerusalem artichoke (Helianthus tuberosus)mitochondria. Biochem J 194: 487–495

Møller IM, Liden AC, Ericson I, Gardestrom P (1987)Isolation of submitochondrial particles with differentpolarities. Method Enzymol 148: 442–453

Møller IM, Jensen PE, Hansson A (2007) Oxidativemodifications to cellular components in plants. AnnuRev Plant Biol 58: 459–481

Morohashi Y, Bewley JD, Yeung EC (1981) Biogenesis ofmitochondria in imbibed peanut cotyledons – influenceof the axis. J Exp Bot 32: 605–613

Motulsky HJ, Christopoulos A (2003) Fitting Models toBiological Data Using Linear and Nonlinear Regression.A Practical Guide to Curve Fitting. Graphpad SoftwareInc., San Diego, CA. Available at http://www.graphpad.com (accessed September 2006)

Nash D, Wiskich DT (1983) Properties of substantiallychlorophyll-free pea leaf mitochondria prepared bysucrose density gradient separation. Plant Physiol 71:627–634

Neuburger M (1985) Preparation of plant mitochondria,criteria for assessment of mitochondrial integrity andpurity, survival in vitro. In: Douce R, Day DA (eds)Higher Plant Cell Respiration: Encyclopedia of PlantPhysiology (New Series, Vol 18). Springer-Verlag,Berlin, pp 7–24

Oliver MJ (1996) Desiccation tolerance in vegetative plantcells. Physiol Plant 97: 779–787

Oliver MJ, Tuba Z, Mishler BD (2000) The evolution ofvegetative desiccation tolerance in land plants. PlantEcol 151: 85–100

Oliver MJ, Velten J, Mishler BD (2005) Desiccationtolerance in bryophytes: a reflection of the primitivestrategy for plant survival in dehydrating habitats? IntegrComp Biol 45: 788–799

Pammenter NW, Berjak P (1999) A review of recalcitrantseed physiology in relation to desiccation tolerancemechanisms. Seed Sci Res 9: 13–37

Pastore D, Trono D, Laus MN, Di Fonzo N, Flagella Z(2007) Possible plant mitochondria involvement in celladaptation to drought stress: a case study: durum wheatmitochondria. J Exp Bot 58: 195–210

Pical C, Fredlund KM, Petit PX, Sommarin M, Møller IM(1993) The outer membrane of plantmitochondria contains a calcium-dependent protein


kinase and multiple phosphoproteins. FEBS Lett 336:347–351

Prasad TK, Anderson MD, Stewart CR (1994) Acclimation,hydrogen-peroxide, and abscisic-acid protectmitochondria against irreversible chilling injury inmaize seedlings. Plant Physiol 105: 619–627

Proctor MCF, Oliver MJ, Wood AJ, Alpert P, Stark LR,Cleavitt NL, Mishler BD (2007) Desiccation tolerance inbryophytes: a review. Bryologist 110: 595–621

Rachmilevitch S, Xu Y, Gonzalez-Meler MA, Huang BR,Lambers H (2007) Cytochrome and alternative pathwayactivity in roots of thermal and non-thermal Agrostisspecies in response to high soil temperature. PhysiolPlant 129: 163–174

Rascio N, La Rocca N (2005) Resurrection plants: thepuzzle of surviving extreme vegetative desiccation. CritRev Plant Sci 24: 209–225

Reisdorph NA, Koster KL (1999) Progressive loss ofdesiccation tolerance in germinating pea (Pisumsativum) seeds. Physiol Plant 105: 266–271

Ribas-Carbo M, Taylor NL, Giles L, Busquets S,Finnegan PM, Day DA, Lambers H, Medrano H,Berry JA, Flexas J (2005) Effects of water stress onrespiration in soybean leaves. Plant Physiol 139:466–473

Roach T, Beckett RP, Minibayeva FV, Colville L,Whitaker C, Chen HY, Bailly C, Kranner I (2010)Extracellular superoxide production, viability and redoxpoise in response to desiccation in recalcitrant Castaneasativa seeds. Plant Cell Environ 33: 59–75

Sanders D, Pelloux J, Brownlee C, Harper JF (2002)Calcium at the crossroads of signaling. Plant Cell 14:S401–S417

Senaratna T, McKersie BD (1983) Dehydration injury ingerminating soybean (Glycine Max Merr, L.) seeds.Plant Physiol 72: 620–624

Simons BH, Millenaar FF, Mulder L, Van Loon LC,Lambers H (1999) Enhanced expression and activationof the alternative oxidase during infection of Arabidopsiswith Pseudomonas syringae pv tomato. Plant Physiol120: 529–538

Smiri M, Chaoui A, El Ferjani E (2009) Respiratorymetabolism in the embryonic axis of germinatingpea seed exposed to cadmium. J Plant Physiol 166:259–269

Song SQ, Jiang CH, Chen RZ, Fu JR, Engelmann F (2003)Dehydration damage and its repair in imbibed soybean(Glycine max) seeds. In: Nicolas G, Bradford KJ, ComeD, Prechard HW (eds) The Biology of Seeds: RecentResearch Advances. CABI Publishing, Oxon,pp 355–370

Song SQ, Tian MH, Kan J, Cheng HY (2009) The responsedifference of mitochondria in recalcitrant Antiaris

toxicaria axes and orthodox Zea mays embryos todehydration injury. J Integr Plant Biol 51: 646–653

Stupnikova I, Benamar A, Tolleter D, Grelet J, Borovskii G,Dorne AJ, Macherel D (2006) Pea seed mitochondriaare endowed with a remarkable tolerance to extremephysiological temperatures. Plant Physiol 140: 326–335

Sun WQ (1999) Desiccation sensitivity of recalcitrantseeds and germinated orthodox seeds: can germinatedorthodox seeds serve as a model system for studies ofrecalcitrance? In Marzalina M, Khoo KC, Jayanthi N,Tsan FY, Krishnapillay B (eds) Proceedings of IUFROSeed Symposium 1998: Recalcitrant Seeds. FRIM, KualaLumpur, Malaysia, pp 29–42

Vertucci CW, Farrant JM (1995) Acquisition and loss ofdesiccation tolerance. In: Kigel J, Galili G (eds) SeedDevelopment and Germination. Marcel Dekker Inc.,New York, pp 149–184

Wang WQ, Song SQ, Li SH, Gan YY, Wu JH, Cheng HY(2009) Quantitative description of the effect ofstratification on dormancy release of grape seeds inresponse to various temperatures and water contents. JExp Bot 60: 3397–3406

Watanabe CK, Hachiya T, Terashima I, Noguchi K (2008)The lack of alternative oxidase at low temperature leadsto a disruption of the balance in carbon and nitrogenmetabolism, and to an up-regulation of antioxidantdefence systems in Arabidopsis thaliana leaves. PlantCell Environ 31: 1190–1202

White PJ, Broadley MR (2003) Calcium in plants. Ann Bot92: 487–511

Wu JH, Wang WQ, Song SQ, Cheng HY (2009) Reactiveoxygen species scavenging enzymes and down-adjustment of metabolism level in mitochondriaassociated with desiccation tolerance acquisition ofmaize embryo. J Integr Plant Biol 51: 638–645

Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Fig. S1. Correlation of latency of crude mitochondrialCCO 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 peaembryo axis mitochondria during (re-)imbibition.

Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.

Edited by P. Gardestrom


the role of recovery of mitochondrial ... -...

Documents