78. Cellular and Metabolic Events Associated withDehydration of Recalcitrant Araucaria angustifoliaEmbryos

F. CORBINEAU, L. SALMEN ESPINDOLA, D. VINEL and D. COMEPhysiologie Vegetale Appliquee, Universite Pierre et Marie Curie, Tour 53, ler etage, 4 place Jussieu,75252 Paris cedex 05, France

Abstract

The aim of the present work was to investigate the sequence of some cellular andmetabolic events occurring in the embryonic axes, which might be related to theloss of viability of recalcitrant Araucaria angustifolia embryos during dehydrationin the open air at 25°C and 55% relative humidity. The decreases in the ability forprotein synthesis and in the capacity to convert l-atninocyclopropane I-carboxylicacid to ethylene, which were observed respectively at 0.5 and 1.5 h ofdehydration,were very early indicators of deterioration. A high increase in· leakage ofelectrolytes, which indicated a deterioration of cell membrane properties, wasobserved by the third-fourth h ofdesiccation. ATP content and energy charge alsorapidly decreased during dehydration. However, energy charge cannot be a goodmarker of damage, since reimbibition of embryos restored its value close to thatmeasured in non-dehydrated axes.

Introduction

Seed dehydration, which results in reduced metabolism, is the normal terminalevent in the development of many seeds (Bewley and Black, 1994). Such seedsthat can be stored in the dry state are called orthodox (Roberts, 1973). Seeds ofseveral species have been termed recalcitrant (Roberts, 1973) because, asopposed to orthodox seeds, they are high in moisture content and cannotwithstand intensive desiccation. This applies to various large-seeded hardwoods(e.g. Castanea, Quercus, Juglans) and numerous important tropical and sub­tropical trees (King and Roberts, 1979; Chin and Roberts, 1980).Unfortunately, while there is considerable information on biochemical injuryduring loss of viability of orthodox seeds (Priestley, 1986), only few dataconcern the metabolic damage associated with dehydration injury of recalci­trant seeds. The aim of the present work was to precise the cellular andmetabolic consequences of desiccation in Araucaria angustifolia embryos, whichare typically recalcitrant (Salmen Espindola et al., 1994), and to determine thepossible sequence of these events occurring in the embryonic axis.

R.H. Ellis, M. Black, A.J. Murdoch, T.D. Hong (eds.), Basic and Applied Aspects ofSeed Biology,pp. 715-721.© 1997 Kluwer Academic Publishers, Dordrecht.

716 F. Corbineau, L. Salmen Espindola, D. Vinel and D. Come

Materials and Methods

Plant Material and Dehydration Method

Experiments were carried out with embryos isolated from freshly harvestedAraucaria angustifolia seeds collected in the southern part of Brazil. Embryoviability was estimated by the germination percentages obtained after 7 days (d)at 25°C as described by Salmen Espindola et al. (1994).In order to study the effects of dehydration on embryo viability and on

metabolic damage induced in the embryonic axis by desiccation, isolatedembryos were placed for various periods in the open air at 25°C and 55%relative humidity. Moisture content of whole embryos or embryonic axes (15replicates) was calculated ona dry weight basis. Dry weight was obtained byoven drying the embryos or the embryonic axes at 105°C for 3 d.

Electrolyte Leakage Measurements

Solute leakage was determined by placing 4 embryonic axes in 10 ml distilledwater at 25°C and measuring the conductivity of the medium with a K 220CONSORT conductimeter after 2 h of soaking. Results are expressed aspercentages of the total leakage from axes boiled for 5 min in water. Theycorrespond to the means of 4 measurements ±So.

Measurement ofACC Conversion to Ethylene

The conversion of l-aminocyclopropane I-carboxylic acid (ACC) to ethylenewas measured by placing 3 embryonic axes in tightly closed 15-ml flaskscontaining 0.5 ml ACC solution (1 mM) in water. After 24 h incubation at30°C, a I-ml gas sample was taken from each flask and injected into a gaschromatograph (type 330, Girde1-France) equipped with a flame ionizationdetector and an activated alumina column for ethylene determination. Resultsare the means of 3 measurements ±SD, and are expressed as the percentages ofethylene produced by axes from freshly isolated (non-dehydrated) embryos(4.5±2.1 n1 per h and per axis).

Adenosine Phosphate Assays

Adenosine phosphates were extracted from one isolated axis according toOlempska-Beer and Bautz Freeze (1984). ATP, ADP and AMP contents of theextracts were measured using the bioluminescence method (Strehler and Totter,1952) with a pico-ATP biophotometer. ADP and AMP were transformed intoATP as described by Saglio et al. (1979). The results obtained are expressed innmol per g dry matter and are the means of 5-8 measurements ±SD.The energy charge was calculated by the ratio (ATP+0.5 ADP)/(ATP+ADP+AMP) defined by Atkinson (1968).

Dehydration ofrecalcitrant Araucaria angustifolia embryos 717

Measurement oft 5SjMethionine Incorporation in Total Protein

Four embryonic axes were sterilized with 1% calcium hypochlorite for 10 min.Mter washing in sterilized water, they were incubated in 200 ~ 0.6 kBq ~-1

eSS]methionine (Amersham, UK) for 2 h. At the end of the in vivo labellingperiod, organs were washed with sterilized water and their proteins wereextracted as described by Salmen Espindola et al. (1994). Methionine incorpora­tion into total protein was expressed as percentage of the [3sS]methionineuptake by the embryonic axes. Results presented correspond to the means of 3measurements ±SD and to the percentages of methionine incorporation byaxes from freshly isolated (non-dehydrated) embryos (21.9±3.7 % uptake).

Results

Sensitivity ofEmbryos to Desiccation

The mean moisture content of freshly isolated (non-dehydrated) embryos wasabout 120% (dry weight basis). It decreased rapidly during desiccation, whichresulted in the loss of embryo viability (Fig. 1). Around 50% of the embryoswere dead when their moisture content had fallen by about 60--65%. The criticalmoisture content at which viability was completely lost was around 25-30%.

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Figure 1. Effects of duration of desiccation on moisture content (1) and viability (2) of embryos.Means of 3 replicates ±SD (moisture content) and of 2 replicates (viability)

718 F. Corbineau, L. Salmen Espindola, D. Vinel and D. Come

Electrolyte Leakage

Electrolyte leakage from embryonic axes increased progressively with decreas­ing moisture content (Fig. 2, curve 1). However, it was not a good indication ofgermination ability ofembryos since it significantly increasedwhen the moisturecontent fell to about 40%, i.e. when about 70% of the embryos had becomeunable to germinate (cf Fig. 1).

Conversion ofACC to Ethylene

Desiccation of embryos was also associated with a decrease in the ability of theembryonic axes to convert ACC to ethylene (Fig. 2, curve 2). This decreaseoccurred when the moisture content ofaxes reached less than 70%, i.e. after only1 h of dehydration. Below 50% moisture content, the ACC-dependent ethyleneproduction was almost nil.

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Figure 2. Effects of duration of desiccation of embryos and of corresponding moisture content ofembryonic axes on electrolyte leakage (I), conversion of ACC to ethylene (2) and [3SS]methionineincorporation in total protein (3) by embryonic axes. Means of 3 (ethylene production and[3sS]methionine incorporation) or 4 (leakage) replicates ±SD

Dehydration a/recalcitrant Araucaria angustifolia embryos 719

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Moisture content (% dry weight)

Figure 3. Effects of duration of desiccation of embryos and of corresponding moisture content ofembryonic axes on ATP (1), ADP (2) and AMP (3) contents of embryonic axes, and on energycharge (4). Means of 5 to 8 measurements ±SD

Incorporation of(5S]Methionine in Total Protein

Dehydration resulted in a decrease in incorporation of eSS]methionine in totalprotein in the embryonic axes (Fig. 2, curve 3), although the methionine uptakewas not strongly reduced (data not shown). A small decrease in moisturecontent was sufficient to inhibit protein synthesis. The incorporation ofmethionine in proteins was reduced by about 25% and 75% when the moisturecontent fell to about 95% and 40%, respectively.

Energy Metabolism

The adenylate pool (ATP+ADP+AMP) of the embryonic axes was significantlyreduced only by the sixth day of desiccation (data not shown), i.e. when allembryos had become unable to germinate (cf Fig. I). But dehydration induced aclear decrease in ATP and ADP levels and an increase in AMP content (Fig. 3).These changes in adenine nucleotide levels resulted in a decline in the energycharge (Fig. 4). These changes in energy metabolism were noticeable by the firsthour of desiccation.

720 F. Corbineau, L. Salmen Espindola, D. Vinel and D. Come

Discussion and Conclusion

The moisture content at which viability of Araucaria angustifolia embryos iscompletely lost is about 25-30%. It is similar to that reported by Corbineau andCome (1986, 1988) for Mangifera indica, Symphonia globulifera and Hopeaodorata seeds and by Fu et al. (1990) for Litchi chinensis and Euphorbia longanseeds.Embryos of freshly harvested Araucaria angustifolia seeds are metabolicallyactive, though cytological observations by Salmen Espindola et al. (1994) haveshown that most of the nuclei of the meristem zone of the radicle are quiescent.These embryos are characterized by high protein synthesis and energy charge.Dehydration results in decreases in various metabolic activities, among whichloss of the ability to incorporate methionine in proteins is one of the earliestindicators of cell deterioration. Desiccation also induces an increase in electro­lyte leakage and a decrease in the ability to convert ACC into ethylene. Theseresults are consistent with the concept that cell membranes are progressivelydamaged by dehydration. Similar increase in solute leakage during desiccationwas observed in recalcitrant seeds of Quercus robur by Poulsen and Eriksen(1992), silver maple and areca palm by Becwar et al. (1982), and Landolphiakirkii by Pammenter et al. (1991). Decrease in the ability to oxidize ACC intoethylene is also a good indication ofmembrane injury since the in vivo activity ofACC oxidase is known to depend on membrane integrity (Odawara et al., 1977).Our results show that the decline in ACC oxidase activity is an earlier indicatorof membrane deterioration than the increase in electrolyte leakage. As inorthodox seeds (Bewley, 1979), decreases in respiratory activity (SalmenEspindola et al., 1994) and in ATP level and energy charge are associated withdesiccation of Araucaria angustifolia embryos. However, reimbibition of em­bryos restores the energy charge to a value close to that measured in non­dehydrated embryos (data not shown). As the oxygen uptake (Salmen Espindolaet al., 1994), the energy charge cannot therefore be considered as a marker ofdamage induced by desiccation.Our results show that a sequence of irreversible cellular and metabolicdamage is associated with desiccation ofAraucaria angustifolia embryonic axes,but it is difficult to know whether it is a cause or consequence of the loss ofviability. Oxidative processes and free radical accumulation will be the subjectof further investigations, since they are usually supposed to be involved in celldeterioration during dehydration (Bewley, 1979; Leprince et al., 1990; Hendry etal., 1992). In this point, study of the possibility of repairing injury uponrehydration through free radical- and peroxide-scavenging enzyme activitiesand/or antioxidant compounds might be required.

Dehydration ofrecalcitrant Araucaria angustifolia embryos 721

References

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Chin, H.F. and Roberts, E.H. 1980. Recalcitrant Crop Seeds, pp. 152. Kuala Lumpur: TropicalPress SDN.

Corbineau, F. and Come, D. 1986. Seed Science and Technology 14: 585-591.Corbineau, F. and Come, D. 1988. Seed Science and Technology 16: 97-103.Fu, lR., Zhang, B.Z.,Wang, x.F., Qiao, Y.Z. and Huang, X.L. 1990. Seed Science and Technology18: 743-754.

Hendry, GA.F., Finch-Savage, W.E., Thorpe, P.e., Atherton, N.M., Buckland, S., Nilson, K.A.and Seel, W.E. 1992. New Phytologist 122: 273-279.

King, M.W. and Roberts, E.H. 1979. The storage of recalcitrant seeds. Achievements and possibleapproaches, pp. 96. Rome: International Board for Plant Genetic Resources.

Leprince, 0., Deltour, R., Thorpe, P.e., Atherton, N.M. and Hendry, G.A.F. 1990. NewPhytologist 116: 573-580.

Odawara, S.A., Watanabe, H. and Imaseki, H. 1977. Plant Physiology 18: 569-575.Olempska-Beer, Z. and Bautz Freeze, E. 1984. Analytical Biochemistry 140: 236-245.Pammenter, N.W., Vertucci, C. and Berjak, P. 1991. Plant Physiology 96: 1093-1098.Poulsen, K.M. and Eriksen, E.N. 1992. Seed Science Research 2: 215-221.Priestley, D.A. 1986. Seed aging. Implications for seed storage and persistance in the soil, pp. 304.Ithaca, New York: Cornell University Press.

Roberts, E.H. 1973. Seed Science and Technology 1: 499-514.Saglio, P.H., Daniels, M.l and Pradet, A. 1979. Journal ofGeneral Microbiology 110: 13-20.Salmen Espindola, L., Noin, M., Corbineau, F. and Come, D. 1994. Seed Science Research 4: 193-201.

Strehler, B.L. and Totter, IR. 1952. Archives ofBiochemistry and Biophysics 40: 28-40.