proteins of cotyledons of mature horse chestnut seeds

1021-4437/01/4801- $25.00 © 2001

MAIK “Nauka

/Interperiodica”0001

Russian Journal of Plant Physiology, Vol. 48, No. 1, 2001, pp. 1–11. Translated from Fiziologiya Rastenii, Vol. 48, No. 1, 2001, pp. 5–17.Original Russian Text Copyright © 2001 by Gumilevskaya, Azarkovich, Komarova, Obroucheva.

INTRODUCTION

As distinct from quiescent seeds, mature viableseeds at deep dormancy cannot germinate even underfavorable conditions. In many species, dormancy canbe overcome by a long-term treatment of seeds with alow temperature (2–10

°

C), or so-called cold stratifica-tion.

Seeds differ not only in the dormancy type but alsoin their relation to desiccation. Orthodox seeds toleratewater loss to about 10% water content without a loss ofviability. However, some seeds (so-called recalcitrantseeds) cannot survive desiccation below the level of30–50% water content [1–3]. The causes of desiccationintolerance in recalcitrant seeds and the mechanisms oforthodox seed tolerance are studied actively. The seedsof both types can exhibit deep dormancy.

The nature of deep dormancy maintenance andrelease was studied from various points of view [4, 5].However, no unique concept was developed, partiallybecause of the diversity of dormancy types [6, 7]. Thecauses for seed transition to the state of deep dormancy,its biochemical mechanisms, as well as the mecha-

nisms of dormancy breaking in orthodox and recalci-trant seeds remain obscure. Nevertheless, dormancyrelease and germination triggering were shown to bedifferent processes: the first one is under hormonal con-trol, whereas the hydration level of axial organs triggersgermination [8]. The preparation for cell elongation inaxial organs and the initiation of growth occur similarlyin seeds of any type of dormancy [9].

Horse chestnut seeds are recalcitrant: they retain ahigh level of hydration (above 50%) after falling fromtrees and do not tolerate desiccation [10]. There areconvincing data that chestnut seeds enter deep dor-mancy [9]. Under favorable water supply and tempera-ture (27

°

C) conditions, freshly-fallen and shortly strat-ified seeds exhibited a low germinability (only 15% ofseeds germinated during the month following the onsetof stratification) [9]. We observed that the length of iso-lated axes also remained almost unchanged during amonth of seed incubation in water at 20

°

C. Thus, horsechestnut seeds were in the state of deep dormancy dur-ing the first month of cold stratification. With increas-ing duration of stratification, seed capacity to germi-nate under favorable conditions increased and the timerequired for their germination was reduced [9]; that is,seeds began to gradually release from dormancy. It took

Proteins of Axial Organs of Dormant and Germinating Horse Chestnut Seeds:

1. General Characterization

N. A. Gumilevskaya*, M. I. Azarkovich**, M. E. Komarova**, and N. V. Obroucheva**

* Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax: 7 (095) 954-2732** Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;

fax: 7 (095) 977-8018; e-mail: [email protected]

Received July 7, 2000

Abstract

—This is the first characterization of proteins from axial organs of recalcitrant horse chestnut seedsduring deep dormancy, dormancy release, and germination. We demonstrated that, during the entire period ofcold stratification, axial organs were enriched in easily soluble albumin-like proteins and almost devoid of glob-ulins. About 80% of the total protein was found in the cytosol. Approximately one third of cytosolic proteinswere heat-stable polypeptides, which were major components of total proteins. Heat-stable proteins comprisedthree groups of polypeptides with mol wts of 52–54, 24–25, and 6–12 kD with a predominance of low-molec-ular-weight proteins. The polypeptide patterns of heat-stable and thermolabile proteins differed strikingly.Heat-stable proteins accumulated in axes during the late seed maturation, comprising more than 30% of thetotal protein in axes of mature seeds. The polypeptide patterns of the total protein of axial organs and its partic-ular fractions did not change in the course of seed dormancy and release. At early germination, the content ofheat-stable proteins in axes decreased and their polypeptide pattern changed both in the cytosol and cell struc-tures. We believe that at least some heat-stable proteins can function as storage proteins in the axes. Localizationof storage proteins in the cells of axial organs and the role of heat-stable proteins in recalcitrant seeds are dis-cussed.

Key words: Aesculus hyppocastanum - seeds - proteins - dormancy - germination - heat-stable proteins

Abbreviations

: BA—benzyladenine; PMSF—phenylmethylsulfo-nyl fluoride; TCA—trichloroacetic acid.

2

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

Vol. 48

No. 1

2001

GUMILEVSKAYA

et al

.

about 16 weeks to break dormancy. Therefore, betweenthe sixth and tenth weeks of chilling, dormancy releaseoccurred. Later, seeds acquired the capacity for rapid(2–3 days) germination at 27

°

C; that is, they were nolonger in the dormant state [9]. It is of interest that, after17 weeks of stratification, seeds began to germinateeven while remaining in moistened sand at 4

°

C, i.e.,without transfer to the optimum (27

°

) temperature.

To elucidate the molecular processes responsible fordormancy maintenance, dormancy release, and the ger-mination of horse chestnut seeds, it may be promisingto investigate the state, properties, and metabolism ofproteins in axial organs as dependent on the progress ofand release from dormancy. Until now, these problemswere almost not studied with regard to recalcitrantseeds. At the same time, a specific feature of theseseeds, viz., the high water content during dormancy,must influence protein storage and mobilization in theaxes. The objective of this work was to characterize thetotal protein of axial organs in horse chestnut seeds dur-ing deep dormancy, dormancy release, and germina-tion. Because of extremely scarce information abouthorse-chestnut seed proteins, we started with proteinsolubility, their polypeptide composition, intracellulardistribution, and thermotolerance in axes and also com-pared these proteins with those in cotyledons.

MATERIALS AND METHODS

Plant material.

Freshly-fallen seeds of horse chest-nut (

Aesculus hyppocastanum

L.) trees were gatheredin 1998 and 1999 in the arboretum in Mogilev(Belarus) and stored in moistened sand at 4

°

C in dark-ness. Under these conditions, seeds began to activelygerminate after 17–19 weeks. We regularly sampled,seeds for the analysis of the polypeptide composition intheir axial organs. For this purpose, seeds were surface-sterilized with 2% calcium hypochlorite for 2 min,washed with running tap water for 1 h, then axialorgans were excised manually, frozen, and stored at

−

20

°

C. In some cases, we analyzed pieces of cotyle-dons that were also frozen and stored at –20

°

C.

In some experiments, seeds were imbibed at 27

°

C inwater and 50 mg/l BA (stimulation of dormancy release[9]) for 60 or 42 h after 6 or 10 weeks of stratification,respectively.

Subcellular fractionation of the homogenate

ofaxial organs was performed by differential centrifuga-tion. All procedures were carried out in the cold. Eightembryo axes were ground first with a mortar and pestleand then in a glass homogenizer with 15 ml of 0.05 MTris–HCl buffer, pH 7.2, containing 0.25 M sucrose,0.01 M Mg-acetate, 0.025 M KCl, and 1 mM PMSF.The homogenate was squeezed through 4 layers ofcheesecloth and centrifuged at 1000

g

for 10 min toremove debris and starch. The volume of the superna-tant (crude cell extract) was adjusted to 15 ml. Aliquots(usually 2 ml) were taken for preparation of protein

samples for electrophoresis, and the extract was centri-fuged at 20 000

g

for 20 min. The pellet was resus-pended in the homogenization buffer, and the suspen-sion was centrifuged at 20 000

g

for 10 min. The washedpellet obtained (the fraction of subcellular structures)was used for electrophoresis. The supernatant (postmi-tochondrial cell extract) was filtered through the cottoncloth, samples for electrophoresis were taken (usually2–2.5 ml), and the remaining extract was heated at75

°

C in a water bath for 5 min for discrimination ofheat-stable and thermolabile proteins. After 1 or 2 minof heating, the solution became turbid, and then anabundant flaky precipitate formed. Coagulated proteinswere recovered by centrifugation at 6000

g

for 10 min,and the pellet was used for the electrophoresis of ther-molabile proteins. The supernatant obtained after theremoval of thermolabile proteins was taken for electro-phoresis of heat-stable proteins.

Fractionation of salt-soluble proteins from axialorgans.

Eight axes were ground with a mortar and pes-tle and then in a glass homogenizer with 10 ml of ahigh-salt buffer containing 0.1 M Tris, pH 8.0, 1 MNaCl, and 1 mM PMSF. Proteins were extracted in anice bath for 2 h with continuous stirring. The extractwas centrifuged at 20 000

g

for 30 min. The supernatant(salt extract) was dialyzed against distilled water for48 h in the cold with five water replacements of 2 l each.The dialyzate was centrifuged at 20 000

g

for 20 min;the supernatant was used for the analysis of water-sol-uble proteins (albumins). The pellet was washed withdistilled water and used for the electrophoresis of glob-ulins.

Preparation of protein samples for SDS-electro-phoresis.

Proteins were precipitated and washed withcold 7% TCA; TCA was removed by three washingswith cold acetone. After acetone removal, proteins weresolubilized in a sample buffer containing 62.5 mM Tris,pH 6.8, 8 M urea, 2% SDS, 5% 2-mercaptoethanol,1 mM PMSF, and bromphenol blue. Samples wereheated at 100

°

C for 2–4 min, cooled, and centrifuged at10 000

g

for 10 min to remove insoluble material. Thesupernatant was used for SDS-electrophoresis. Equalamounts of protein (usually 40–50

µ

g) were loaded ongel lanes.

SDS-electrophoresis

was carried out under reducingdenaturing conditions in the system used by O’Farrell[11] for the second electrophoretic direction, asdescribed earlier [12]. Electrophoresis was performedin gradient (10–20%) slab gels at pH 8.8 in the separat-ing gel; 6% PAAG, pH 6.8, was used as the concentrat-ing gel. Tris–glycine buffer, pH 8.3, containing 1%SDS served as an electrode buffer. Gels were fixed andstained overnight with 0.1% Coomassie R-250 in themixture of methanol, acetic acid, and water (5 : 1 : 4).After destaining, gels were photographed and dried.For the determination of polypeptide mol wts, a set ofstandard proteins from Sigma (United States) was used:myosin, 205 kD;

β

-galactosidase, 116 kD;

β

-phospho-


Vol. 48

No. 1

2001

PROTEINS OF AXIAL ORGANS 3

rylase, 97 kD; bovine serum albumin, 66 kD; ovalbu-min, 45 kD; glycerophosphate dehydrogenase, 36 kD;carbonic anhydrase, 30 kD; trypsin, 24 kD; trypsininhibitor, 20 kD;

β

-lactalbumin, 14 kD; lysozyme, 12 kD;and aprotenin, 6.5kD.

Protein estimation

was performed by method [13]with minor modifications [12]. Samples were put onWhatman filter paper, fixed with cold 10% TCA,washed first with cold, then hot 10% TCA, a mixture ofethanol and ethyl ether (1 : 1), and finally with ethylether. Paper was stained with amido black and thendestained with 10% acetic acid. Protein-bound dye waseluted and measured at 620 nm. For protein determina-tion, bovine serum albumin was used for the standardcurve. The content of protein in the homogenate frac-tions was calculated from the protein content in sam-ples for electrophoresis. All data are means from threereplicates with three recordings in each.

Reagents

. Tris, urea, and acrylamide were recrystal-lized; glycine, SDS, mol wt markers, and CoomassieR-250 were obtained from Sigma (United States);PMSF from Serva (Germany); all other reagents wereproduced domestically and were of analytical grade.

RESULTS

Protein extraction and fractionation.

There arediverse methods for the extraction of the total proteinfrom tissues. Any method should meet the followingrequirements: it must ensure the completeness ofextraction, minimize the possibility of proteolytic deg-radation during protein isolation, and provide for stablepreparations and reproducible results. We tested threemethods for producing preparations of the total proteinfrom horse chestnut axes and compared them: (1) tissuefixation with cold TCA, its removal with cold acetone,protein extraction with law-salt Tris buffer, pH 6.8,containing urea, SDS, and mercaptoethanol (samplebuffer) at 100

°

C; (2) preparation of the crude cellextract by tissue homogenization in 0.25 M sucrose ina low-ionic-strength buffer, protein precipitation withTCA with subsequent extraction by the sample buffer;and (3) protein salt extraction and precipitation withTCA. Earlier, we successfully applied all three meth-ods for the characterization of pea seeds [14]. However,when the first method was applied to horse chestnutseeds, we encountered some difficulties. Thus, sampleheating at 100

°

C resulted in the solution thickening,evidently due to the high content of starch. During sub-sequent centrifugation, the bulk of material was lost. Inaddition, in most cases, some compounds from chest-nut tissues evidently acidified the sample buffer, sincebromphenol blue changed to yellow-blue or green. Itwas also suspicious that only a few proteins with molwts above 50 kD were seen on electrophoregrams ofsuch protein preparations (Fig. 1, lane

1)

. In addition,the amount of extracted proteins was low, the resultswere poorly reproducible, and the amount of loadedprotein did not correspond to that revealed in the

stained gel. The samples prepared for analysis wereunstable and spoiled after several days of storagealthough they had been frozen. It seemed that we couldnot correctly quantify protein in the samples.

It is conceivable that, with this method for materialtreatment, buffer acidification in the protein sampleresults in the isoelectric precipitation of the protein orits incomplete denaturation and solubilization and (or)a nonquantitative formation of SDS–protein complexesin spite of the combined application of such denaturingagents as SDS and urea. To overcome these difficulties,we tried to elucidate what cell fraction contained com-pounds interfering with the appropriate electrophoreticanalysis. When the homogenate from axial organs wassubjected to differential centrifugation, it turned outthat the 1000

g

centrifugate (the fraction devoid of cellnuclei) was suitable for electrophoretic analysis of totalprotein from the axes. We obtained stable preparations,which could be stored for at least two months, with high-molecular-weight (>50 kD) polypeptides seen on thechromatograms, quite reproducible electrophoretic pat-terns, and a much higher yield of extracted proteins (1.9–2.2 vs. 0.2–0.3 mg/axis) (Fig. 1, lane

2

). When we triedto extract protein from the cell debris remaining on thecheesecloth filter, we encountered similar difficulties asafter axial organ extraction. The material was fixedwith TCA, washed with acetone, and suspended in thesample buffer. As a result, buffer color changed fromviolet to bright yellow-green and the sample substan-tially thickened during heating. After removal of theabundant green pellet, the content of soluble protein didnot exceed 0.1 mg/axis. The fraction enriched with cellfragments, nuclei, starch grains, and (or) other polysac-charides (1000

g

pellet) also contained a very lowamount of protein and became thickened during heat-ing. However, it did not acidify the buffer, and the sam-ple was bright blue. Thus, to obtain an appropriate sam-ple for the analysis of horse-chestnut axis protein, itwas necessary to remove cell debris and the materialsedimented at 1000

g

for 10 min, which contained abulk of starch and other polysaccharides. Since proteinin the 1000

g

pellet comprised only 4–5% of the proteinin the 1000

g

supernatant, we neglected this fraction andconsidered protein in the 1000

g

supernatant as the totalprotein of axial organs.

Along with total proteins, we analyzed proteins inthe fraction of cell structures (20 000

g

pellet) and post-mitochondrial supernatant in order to detect additionalpolypeptides and, on the other hand, to characterize theintracellular distribution of proteins.

Thus, we believe that the procedure described abovefor protein extraction and fractionation is the best onefor studying the proteins from horse chestnut axes. Theconditions selected for electrophoresis (discontinuousbuffer system and PAAG gradient) were satisfactory forpolypeptide separation. The total pattern of polypep-tides was stable and well reproducible over the two-year period of the experiment. Therefore, the patterns

4


Vol. 48

No. 1

2001

GUMILEVSKAYA

et al

.

observed reflect a real heterogeneity of polypeptides ofthe total protein from axial organs but do not indicate adegradation by proteases.

Salt extraction was also effective and gave similarpatterns of polypeptides from axial organs (Fig. 1, lane

3

);it was used for the additional characterization of thetotal protein.

Total protein of axial organs at deep dormancy ofseeds (2–5 weeks of stratification).

During deep dor-mancy, more than 80% of the protein of axial organswas represented by soluble proteins in the postmito-chondrial supernatant. Cell organelles (except nuclei)contained 12–15% of the protein (Fig. 2). Cytosolicproteins differed in their tolerance to heating. Heatingat 75

°

C for 5 min resulted in the coagulation of about60% of the total protein (thermolabile proteins), and

almost 30% of protein remained in the solution (heat-stable proteins) (Fig. 2).

One-dimensional SDS-electrophoresis showed thatpolypeptides of the cell extract from axial organs weredistributed along the lane more or less uniformly withinthe range of mol wts from 6 to 100 kD (Fig. 3).Polypeptides with mol wts above 100 kD were seen asminor and hardly outstanding components. Amongmajor polypeptides of both the cytosol and cellorganelles, a complex group with mol wts from 50 to58 kD was seen. Another predominant polypeptide(s)was found in the zone of 24–25 kD, which was charac-teristic of the cytosol. Some moderately abundantpolypeptides were detected: the two groups of low-molecular-weight (8–12 and 18–20 kD) and some high-molecular-weight (74, 84, and 97 kD) polypeptides.These polypeptides were found in both the cytosol andorganelles. In addition, a 42-kD protein was found inthe cytosol and a 32-kD protein in both fractions. Alongwith these major polypeptides from axial organs,numerous minor components, common or specific toeach fraction, were distinguished.

The polypeptide patterns of the cytosol and totalhomogenate were almost identical, indicating theabsence of proteolytic degradation during homogenatefractionation. Most proteins characteristic of cell struc-tures were poorly seen on the chromatograms of the

Fig. 1.

Electrophoregrams of proteins extracted from axialorgans of dormant horse chestnut seeds by various methods.(

1

) Total axial proteins solubilized with the sample buffer(6.25 mM Tris, SDS, urea, and mercaptoethanol) after tissuefixation with TCA; (

2

) proteins of the 1000

g

supernatant(crude cell extract) solubilized with the sample buffer fromTCA-insoluble material; (

3

) salt-soluble proteins of axialorgans solubilized with the sample buffer from TCA-insolublematerial; M—marker proteins; mol wts are indicated in kD.

12

20

24

30

36

48

66

97

116

å

1 2 3

2

3

1

0

10

20

30

40

50

60

70

80

210 17 17 192

CotyledonsPr

otei

n, %

of

prot

ein

in 1

000

g

sup

erna

tant

Weeks

Deep Dormancy Non- Axis elon- Deep

of strati-

dormancy release

dormant dormancy

N P

Axial organs

fication

Fig. 2.

Protein distribution between fractions of the homo-genate of axial organs in the course of seed stratification.P—radicle protrusion; N—no radicle protrusion.(

1

) 20 000

g

pellet (subcellular structures); (

2

) thermolabilecytosolic proteins; (

3

) heat-stable cytosolic proteins.

gation


Vol. 48

No. 1

2001


total protein because the fraction of structures con-tained only a small portion of the total protein or due toa possible transit of proteins from disrupted structuresinto the cytosol. This fraction of organelles contained amajor 52-kD polypeptide and numerous minor compo-nents. Polypeptides with mol wts of 15, 21, 60, 62, and68 kD and diffuse bands within the zone of 75 to 90 kDwere characteristic of this fraction.

Electrophoretic analysis of thermolabile and heat-stable protein fractions of the cytosol showed that mostproteins are thermolabile. Heat-stable proteins werepresented by three groups of polypeptides: 52–54, 24–25, and 6–12 kD (Fig. 3). Some minor componentscommon for these two fractions could result from theirmutual contamination, since these fractions were not

additionally purified. An insufficient resolving capacityof one-dimensional electrophoresis could be anothercause. It is worth noting that all three groups of heat-stable polypeptides represented major components ofthe total protein of axial organs.

Most proteins were efficiently extracted with1 M NaCl, and their electrophoretic pattern was similarto that of the total protein (Fig. 1). However, it was dif-ficult to separate extracted proteins into albumins(water-soluble proteins) and globulins (water-insolubleproteins). After long-term dialysis of the salt extractagainst water, which is a common technique for suchseparation, the dialyzate became only slightly turbid,indicating a low content of globulins in the axial organsof horse chestnut seeds. Moreover, the sum of albumins

Fig. 3.

Electrophoregrams of proteins from axial organs of horse chestnut seeds.I—1000

g

supernatant (crude cell extract); II—20 000

g

pellet (subcellular structures); III—20000

g

supernatant (cytosol); IV—ther-molabile cytosolic proteins; V—heat-stable cytosolic proteins. A and G—albumins and globulins (fractions of total salt-soluble pro-teins from axes of dormant seeds). M—marker proteins; mol wts are indicated in kD.

11697

66

48

36

30

24

20

12

21

15

1286

18192022

25–24

30323437

42

505458

74808497

5452

2524

12–6

Weeksof stratification

2 17 17 17 17 172 2 2 2 2 2

I II III IV V A G

Fractions

å

6


Vol. 48

No. 1

2001

GUMILEVSKAYA

et al

.

and globulins comprised only 50–60% of the salt-extracted protein taken for dialysis. It is evident thatthere was a partial loss of albumins during this proce-dure. Nevertheless, we have shown that the major heat-stable proteins were albumins, similarly to most high-molecular-weight components (74, 78, and 87 kD). Thepolypeptides with mol wts of 32, 37, and 42 kD wereglobulins.

Changes in the polypeptide patterns of axial organsduring dormancy maintenance, release, and subse-quent germination.

We compared seeds after 2 weeks(deep dormancy), 10 weeks (dormancy release), 17 weeks(nondormant seeds with radicles protruded or nonpro-truded), and 19 weeks (germinated seeds) of stratifica-tion. As evident from rough calculations, the content oftotal soluble protein slightly decreased in the course ofstratification. However, during radicle protrusion andsubsequent axis growth, the content of proteinincreased by 15–20% and the axes lengthened to 1.0–1.2 and 1.5–2.0 cm, respectively, as compared to 0.6–0.7 cm in dormant seeds. The protein of cell structurescomprised 12–15% of the total protein (Fig. 2), and thisproportion did not practically change during stratifica-tion. In the cytosol, the relative content of heat-stableproteins decreased from 30 to 20%. This decreasebecame more apparent during radicle protrusion andsubsequent axis growth (to 12 and 7%, respectively).Simultaneously, the fraction of thermolabile proteinsgradually increased from 55 to 70%. Rough calcula-tions of the amounts of thermolabile and heat-stableproteins in the cell extract showed that the level of ther-molabile proteins was practically constant in the courseof stratification but increased considerably during radi-cle protrusion and axis growth. In contrast, the level ofheat-stable proteins was almost halved in the course of

stratification and continued to decline during germina-tion (Fig. 4).

When comparing the composition of axial proteinsin the course of stratification, we could not find any sig-nificant changes in the polypeptide patterns of any stud-ied protein fractions. They seemed similar after 2, 6, 10,and 17 weeks of stratification, that is, in deep dor-mancy, during dormancy release, and immediatelybefore radicle protrusion (see Fig. 3). Imbibition ofstratifying intact seeds at 27

°

C in BA favoring dor-mancy release also did not result in rapid changes in thepolypeptide composition of axial organs prior to germi-nation.

Visible changes appeared only after radicle protru-sion and subsequent growth (Fig. 5) encompassing notonly major but also moderately abundant polypeptides.The content of high-molecular-weight components(74–97 kD, group 1) decreased; a large group of pre-dominant proteins with mol wts from 50 to 58 kD(group 2) became less complex; the amount of majorcomponents with mol wts of 24–25 kD (group 3) andsome small polypeptides (6–12 kD, group 4) slightlydecreased. On the other hand, other polypeptides withmol wts of about 32 kD (group 5) and 15–18 kD (group 6)increased. In the fraction of cell structures (Fig. 5, II),the content of some components from group 1(74 and 92 kD) and dominant proteins from group 2(52–54 kD) was diminished and the components fromgroup 5 (about 32 kD) and group 6 (15–18 kD) becamemore visible. In cytosolic proteins (Fig. 5, III), we alsofound polypeptides (groups 2, 3, and 4) whose contentdecreased during germination. The most pronouncedchanges occurred in the fraction of cytosolic heat-sta-ble proteins (Fig. 5, V). Predominant polypeptides withmol wts of 52–54 kD disappeared almost completely,some components with lesser mol wts appeared, andthe content of major low-molecular-weight proteins (6–12 kD) was substantially decreased. A decrease in thecontent of major 24–25-kD proteins was not clearlyevident in these samples due to their overloading, but itwas visible on the electrophoregrams of cytosolic andtotal proteins. Changes in thermolabile proteins wereslightly noticeable.

Thus, seed germination induced changes mainly inthe heat-stable cytosolic proteins of axial organs; pro-teins of cell structures also contributed to thesechanges, although to a lesser degree.

To better understand the role of embryo axis pro-teins in the physiology of dormancy and germination ofrecalcitrant chestnut seeds, we tried to determine theaccumulation time of the major proteins during seeddevelopment. Since we could not note the time elapsedsince flowering for each seed, we assessed the seed ageindirectly based on the axis sizes, their fresh weight,and the content of protein in them. We gathered chest-nut seeds in Moscow parks on August 21 and Septem-ber 15, 1998. The axes of these immature seeds differedin their average fresh weight (42 and 63 mg/axis), size

0 5 10 15 20 25

2

4

6

8

10

12

14

16

18

Stratification, weeks

Prot

ein,

mg/

8 ax

es

Thermolabile proteins

Heat-stable proteins

Radicleprotrusion

Fig. 4. The content of thermolabile and heat-stable cytosolicproteins in the course of seed stratification.

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 48 No. 1 2001


(0.3 and 0.5–0.6 cm), and the content of protein (0.8and 1.7 mg/axis). Therefore, these two seed lots dif-fered in terms of seed age. We compared total proteinsin the axes of these seeds with thermolabile and heat-stable proteins of mature dormant seeds (2 weeks ofstratification). Figure 6 shows that most of the proteinselectrophoretically similar to the thermolabile proteinsof mature seeds were already present in younger axes,whereas proteins similar to the heat-stable proteins ofmature seeds appeared later, in maturing seeds.

Thus, during the maturation of horse chestnut seedsin cells of axial organs, proteins that accumulated wereresistant to heating, comprised about 30% of the totalprotein, occurred only in the cytosol, and belonged toalbumins. During germination, their content decreasedto 7% of the total protein of the cell extract, and theirpolypeptide patterns changed. To elucidate whetherthese proteins are specific to axial organs and whetherthey can be considered storage proteins of the axes, wecompared proteins of axial organs and cotyledons.

Comparison of proteins from cotyledonary storageparenchyma and axis proteins. Proteins of axial organsand cotyledons have many common characteristics. Inboth cases, the bulk of proteins are detected in the cyto-sol (20 000g supernatant). Proteins of cell structurescomprise only a small portion of the total protein: 15%in axial organs and 5% in cotyledons (Fig. 2).A similarity was found also in the polypeptide patternsof the total protein of axes and cotyledons, both at seedmaturation and deep dormancy (Figs. 6 and 7). In thecytosol of axial organs and cotyledons, there are heat-stable proteins comprising a small number of majorpolypeptides and numerous thermolabile proteins ofmoderate and minor abundance (Figs. 3 and 7). How-ever, while only 25–30% of axial proteins were heat-stable, the portion of such proteins in cotyledonsamounted to 70%. Therefore, it is not surprising thatcotyledonary heat-stable proteins practically deter-mined the pattern of the total protein of the cell extractand cytosol, whereas, in the axial organs, thermolabileproteins determined the pattern of the total protein(Fig. 3). A comparison of heat-stable proteins in axes

Fig. 5. Electrophoregrams of proteins of axial organs of horse chestnut seeds after 17 weeks of stratification (nondormant seeds, noradicle protrusion) and 19 weeks of stratification (germinated seeds).I—1000g supernatant (crude cell extract); II—20 000g pellet (subcellular structures); III—20 000g supernatant (cytosol); V— heat-stable cytosolic proteins. Arabic figures designate protein groups (see the text). M—marker proteins, kD.

12

20

30

36

48

66

97116

å

1

2

5

3

6

4

1

2

5

3

6

2

3

44

3

2

17 17 17 17 19191919

I II III VFractions

Weeksof stratification

8


GUMILEVSKAYA et al.

and cotyledons demonstrated that one of their groups(52–54 kD) was specific to axial organs. Two othergroups of major heat-stable polypeptides (24–25 and6–12 kD) exhibited similar electrophoretic mobilitiesand dominated among the heat-stable proteins in bothcotyledons and axes (Fig. 7).

During seed germination, the content of thermo-labile proteins in cotyledons, as distinct from axes,apparently diminished, since they hardly precipitatedduring heating and were practically lost during a num-ber of subsequent centrifugations. As a result, we couldnot obtain preparations of thermolabile proteins fromcotyledons during radicle protrusion or later, when theaxis grows.

Thus, a preliminary comparative analysis of pro-teins from axial organs and cotyledons revealed a sim-

ilarity along with some differences between them.Heat-stable proteins were the predominant proteins incotyledon storage parenchyma at all developmentalstages: seed maturation, deep dormancy, and germina-tion of stratified seeds. We believe that the heat-stableproteins of cotyledons can be considered the storageproteins of horse chestnut embryos. Since heat-stableproteins in axial organs are very close to those of coty-ledons, we may suggest that they fulfill the role of stor-age proteins in axes.

DISCUSSION

The aim of this work was to characterize the totalprotein of axial organs of recalcitrant mature horsechestnut seeds during deep dormancy, dormancyrelease, and germination; to reveal the effect of cold

Fig. 6. Electrophoregrams of proteins from axial organs andcotyledons of horse chestnut seeds at their maturation anddeep dormancy.(1) Axial organs of dormant seeds (2 weeks of stratification);(2) axial organs of maturing seeds collected on August 21;(3) axial organs of maturing seeds collected on September 15;(4) cotyledonary proteins of maturing seeds collected onSeptember 15. I—1000g supernatant (crude cell extract);IV—thermolabile cytosolic proteins; V—heat-stable cyto-solic proteins. M—marker proteins, kD.

Fig. 7. Electrophoregrams of cotyledonary proteins of dor-mant horse chestnut seeds (2 weeks of stratification).I—1000g supernatant (crude cell extract); II—20 000g pel-let (subcellular structures); III—20 000g supernatant (cyto-sol); IV—thermolabile cytosolic proteins; V—heat-stablecytosolic proteins.

66

36

30

20

12

å

1 2 3 4IV V I IFractions

I II III IV V

Fractions



stratification on protein composition; and to compareproteins in the axes and cotyledons.

The primary properties of proteins in horse chestnutseeds are the following:

(1) Cells of axial organs of mature seeds containextremely low amounts of globulins and are enrichedwith easily soluble albumins during the entire period ofstratification.

(2) A considerable fraction (up to 30%) of easilysoluble proteins extracted with the buffer of low ionicstrength is represented by heat-stable proteins, which atthe same time are the major polypeptides of the totalprotein from axial organs.

(3) In cotyledons, easily soluble heat-stable cytoso-lic proteins represent 70% of the total protein; they areelectrophoretically similar to proteins of axial organs.

The combined data suggest that heat-stable proteinsare the storage proteins of horse chestnut seeds.

It is commonly accepted that the predominant pro-teins in seeds are storage proteins that are deposited inprotein bodies. During differential centrifugation, pro-tein bodies must sediment between 1000 and 20 000g;that is, they must be found in the fraction of cell struc-tures (20 000g pellet). In fact, this fraction containedonly 15% of the total protein of axial organs; neverthe-less, heat-stable proteins were found not here, but asmajor components in the cytosol. The question arises asto where the heat-stable proteins are located in the cellsof axial organs and cotyledons: in the cytosol or in pro-tein bodies that were easily destroyed during homoge-nization and could easily release soluble proteins intothe cytosol fraction. The ultrastructure of horse chest-nut seeds is poorly studied: only two rather contradic-tory publications are known [15, 16]. In both studies, itis emphasized that the ultrastructure of mature horsechestnut seeds resembles that of actively metabolizingtissues, which is not surprising, taking into account thehigh water content in these recalcitrant seeds. One teamof authors [15] observed that, in the course of seeddevelopment, vacuoles in the hypocotyl cells weregradually filled with storage compounds, the endoplas-mic reticulum developed into an extensive net, numer-ous small protein bodies appeared, and large starchgrains accumulated in the amyloplasts. According toother authors [16], the ultrastructure of the axis and cot-yledons was very alike: cells contained a large nucleuswith a nucleolus, numerous mitochondria had a well-developed inner structure, the endoplasmic reticulumwas extensive, and numerous free and bound ribosomesand also amyloplasts with large starch grains wereseen. Dictyosomes of the Golgi apparatus were rare,and their structure was reduced. An important differ-ence was the absence of protein bodies. During theentire period of embryo development, the vacuolesremained unchanged. This is typical of nonproteinseeds, which do not accumulate large amounts of stor-age proteins. Such are the horse chestnut seeds, the

content of protein in which did not exceed 7.5% on adry wt basis [16].

In this context, two considerations are essential.Firstly, the complete destruction of protein bodiesseems unlikely. Secondly, some heat-stable proteins,dehydrins for example, were detected in the cytosol[17]. In addition, according to our data, the fraction ofcell structures contained some proteins whose contentdecreased during germination. It is conceivable thatthis small protein fraction was localized in protein bod-ies, whereas predominant cell proteins were present inthe cytosol. It seems apparent that the occurence andstate of protein bodies in axial organs of mature horsechestnut seeds and the properties of proteins localizedwithin them are not well studied. None of the possibil-ities for storage protein localization outlined above canbe excluded. Protein body isolation in a nonaqueousmedium and the microscopic analysis of sectionsstained for proteins can help to answer this question.

The next point is the influence of stratification onproteins of axial organs. We did not notice any changesin the polypeptide patterns of the total protein. Evi-dently, dormancy maintenance, dormancy release, andseed preparation for germination were not accompa-nied by substantial changes in the composition of thetotal protein or its particular fractions, which we coulddetect by the methods used. Substantial changes wereobserved during radicle protrusion and especially dur-ing early axis growth, when the content of heat-stableproteins and some components from the fraction of cellstructures decreased. These changes can be explainedby proteolytic processes, which supply axial organswith nitrogen compounds during heterotrophic growthafter germination. The results we obtained with horsechestnut seeds differed markedly from the patternobserved with the apple axial organs, in which stratifi-cation induced substantial changes in the polypeptidecomposition of the total protein [18, 19]. The cause ofthis difference is not clear. However, we have toremember that apple seeds belong to the orthodox type,whereas horse chestnut seeds are recalcitrant. It mightbe that storage protein mobilization occurs in these twoplant species at different times and at different rates.

The results concerning the fraction of heat-stableproteins in chestnut embryo axes and cotyledons are ofspecial interest. Heat-stable proteins accumulated dur-ing horse chestnut seed maturation were abundant infreshly fallen mature seeds. They comprised more than30% of the soluble proteins of axial organs and domi-nated (about 70%) among soluble cotyledonary pro-teins. Heat-stable proteins had a characteristic polypep-tide composition. They contained three groups of majorpolypeptides with mol wts of 52–54, 24–25, and 6–12 kD. Low-molecular-weight proteins were especiallyabundant. This pattern was drastically distinct from thatof thermolabile polypeptides. Chestnut thermolabilepolypeptides resemble those from peanut [20], cedar[17], and the barley aleurone layer [21]. The content of

10


GUMILEVSKAYA et al.

axial heat-stable proteins decreased during horse chest-nut seed germination and early seedling growth. It isconceivable that heat-stable proteins fulfill the func-tions of storage proteins and are the source of nitrogenfor seedlings. Such a function was proposed for theheat-stable proteins of the barley aleurone layer [21]and peanut cotyledons [20]. However, heat-stable pro-teins might have some additional functions related totheir extremely high heat resistance, specific aminoacid composition, and high content in the cell [21]. Theinterest in heat-stable proteins is supported by the factthat some proteins of late embryogenesis (LEA pro-teins), including dehydrin-like proteins, belong to thisprotein type, and these proteins are considered a factorof orthodox seed resistance to desiccation [1, 22].These proteins are induced by water deficiency andABA treatment [21, 23]; they can operate as protectantsduring water stress or stabilizers of cell structures dur-ing the progressing desiccation [24]. It had been sup-posed that the sensitivity of recalcitrant seeds to dehy-dration is related to the lack of dehydrins in these seeds[25]. However, dehydrin-like proteins were later foundin the fraction of heat-stable proteins from recalcitrantseeds of woody plants growing in the temperate cli-matic zone, including seeds of horse chestnut [26, 27],but they were not detected in seeds of trees inhabitingdamp tropics. The question arises as to what the func-tion of LEA proteins or dehydrins is in recalcitrantseeds. On the basis of the pattern of dehydrin distribu-tion among various recalcitrant seeds, Farrant et al.[27] concluded the dehydrin-like LEA proteins werepresent in those recalcitrant seeds that were capable ofsurviving partial dehydration during development,were insensitive to low temperature, and containedmuch ABA. These proteins are believed to protect seedsnot only against desiccation, but also against chillingstress [27]. However, dehydrin-like proteins compriseonly a small portion of the heat-stable proteins in recal-citrant seeds. The functions of the major heat-stableproteins in dormant and germinating horse chestnutseeds, the physiological strategy of their high content inthe seed, the causes of their high heat resistance, andtheir responses to low temperature during cold stratifi-cation remain to be studied. The properties and struc-ture of heat-stable proteins, the pattern of their synthe-sis, their distribution among recalcitrant seeds, and sen-sitivity to ABA and heat shock are of great interest.

ACKNOWLEDGMENTS

This work was supported by the Russian Foundationfor Basic Research, project no. 98-04-48630.

REFERENCES

1. Vertucci, C.W. and Farrant, J.M., Acquisition and Lossof Desiccation Tolerance, Seed Development and Germi-nation, Negbi, M. and Kigel, J., Eds., New York: MarcelDekker, 1995, pp. 237–271.

2. Farrant, J.M., Pammenter, N.W., and Berjak, P., Recalci-trance—a Current Assessment, Seed Sci. Technol., 1988,vol. 16, pp. 155–166.

3. Kermode, A.R., Approach to Elucidate the Basis of Des-iccation Tolerance in Seeds, Seed Sci. Res., 1997, vol. 7,pp. 75–95.

4. Bewley, J.D. and Black, M., Physiology and Biochemis-try of Seeds, vol. 2, Berlin: Springer-Verlag, 1982.

5. Bewley, J.D. and Black, M., Seeds: Physiology of Devel-opment and Germination, New York: Plenum, 1994.

6. Nikolaeva, M.G., Seed Dormancy, Fiziologiya semyan(Physiology of Seeds), Prokof’ev, A.A., Ed., Moscow:Nauka, 1982, pp. 125–183.

7. Pozdova, L.M. and Razumova, M.V., Seed Dormancy,Embriologiya tsvetkovykh rastenii. Terminologiya ikontseptsii (Embryology of Phanerogamous Plants: Ter-minology and Concepts), Batygina, T.B., Ed., St. Peters-burg: Mir i Sem’ya-95, 1997, vol. 2, pp. 656–666.

8. Obroucheva, N.V. and Antipova, O.V., The Distinct Con-trolling of Dormancy Release and Germination Com-mencement in Seeds, Dormancy in Plants: from WholePlant Behaviour to Cellular Control, Viémont, J.-D. andCrabbé, J.-J., Eds., Willingford: CAB International,2000, pp. 35–46.

9. Obroucheva, N.V. and Antipova, O.V., Common Physio-logical Mechanisms Prepare Seeds with Different Dor-mancy Types for Germination, Fiziol. Rast. (Moscow),1999, vol. 46, pp. 426–431 (Russ. J. Plant Physiol.,Engl. Transl.).

10. Tompsett, P.B. and Prichard, H.W., The Effect of Chill-ing and Moisture Status on the Germination, DesiccationTolerance and Longevity of Aesculus hyppocastanum L.Seed, Ann. Bot., 1998, vol. 82, pp. 249–261.

11. O’Farrell, P.H., High Resolution Two-Dimensional Elec-trophoresis of Proteins, J. Biol. Chem., 1975, vol. 250,pp. 4007–4021.

12. Skazhennik, M.A., Gumilevskaya, N.A., Kuvaeva, E.B.,and Kretovich, V.L., Electrophoretic Analysis of theComponent Composition of the Total Protein from PeaCotyledons, Prikl. Biokhim. Mikrobiol., 1981, vol. 17,pp. 918–926.

13. Bramhall, S., Noack, N., Wu, M., and Lowenberg, J.,A Simple Colorimetric Method for Determination ofProtein, Anal. Biochem., 1969, vol. 31, pp. 146–148.

14. Gumilevskaya, N.A., Chumikina, L.A., Arabova, L.I.,and Zimin, M.V., The Effect of Heat Shock on the Mobi-lization of Reserve Proteins in the Axial Organs ofImbibing Pisum sativum Embryos, Fiziol. Rast. (Mos-cow), 1993, vol. 40, pp. 110–115 (Sov. Plant Physiol.,Engl. Transl.).

15. Farrant, J.M. and Walters, C., Ultrastructural and Bio-physical Changes in Developing Embryos of Aesculushyppocastanum in Relation to the Acquisition of Toler-ance to Drying, Physiol. Plant., 1998, vol. 104, pp. 513–524.

16. Musatenko, L.I., Generalova, V.M., and Martyn, G.G.,The Physiology of Aesculus hyppocastanum L. SeedDormancy, Ukr. Bot. Zh., 1997, vol. 54, pp. 86–91.

17. Xia, Y.-H. and Kermode, A.R., Analysis to Determine theRole of Embryo Immaturity in Dormancy Maintenanceof Yellow Cedar (Chamaecyperus nootkatensis) Seeds



and Proteins Implicated in Desiccation Tolerance, J. Exp.Bot., 1999, vol. 50, pp. 107–118.

18. Daskalyuk, A.P., Toma, O.K., Yarotskaya, L.V., andNikitina, I.I., Seed Germination and Polypeptide Com-position in Early- and Late-Ripening Apple Cultivars asRelated to Stratification Time, Fiziol. Rast. (Moscow),1996, vol. 463, pp. 574–580 (Russ. J. Plant Physiol.,Engl. Transl.).

19. Eichholtz, D.A., Robintaille, H.A., and Herrmann, K.M.,Protein Changes during the Stratification of Malusdomestica Borkh. Seed, Plant Physiol., 1983, vol. 72,pp. 750–753.

20. Fu, J.R., Yang, X.Q., Jiang, G.X.C., He, J.X., andSong, S.Q., Heat Stable Proteins and Desiccation Toler-ance in Recalcitrant and Orthodox Seeds, Basic andApplied Aspects of Seed Biology, Ellis, R.H., Black, M.,Murdoch, A.J., and Hong, T.D., Eds., Dordrecht: Klu-wer, 1997, pp. 705–713.

21. Jacobsen, J.V. and Shaw, D.C., Heat-Stable Proteins andAbscisic Acid Action in Barley Aleurone Cells, PlantPhysiol., 1999, vol. 91, pp. 1520–1526.

22. Blackman, S.A., Obendorf, R.L., and Leopold, A.C.,Maturation Proteins in Desiccation Tolerance of Devel-

oping Soybean Seeds, Plant Physiol., 1992, vol. 100,pp. 225–230.

23. Galau, G.H., Huges, D.W., and Dure, L. III., AbscisicAcid Induction of Cloned Cotton Late Embryogenesis-Abundant (LEA) mRNAs, Plant Mol. Biol., 1986, vol. 7,pp. 150–170.

24. Lane, B.G., Cellular Desiccation and Hydration: Devel-opmentally Regulated Proteins and the Maturation andGermination of Seed Embryos, FASEB J., 1991, vol. 5,pp. 2893–2901.

25. Farrant, J.M., Berjak, P., and Pammenter, N.W., Proteinsin Development and Germination of a Desiccation Sen-sitive (Recalcitrant Seed) Species, Plant Growth Regul.,1992, vol. 11, pp. 237–265.

26. Finch-Savage, W.E., Pramanik, S.K., and Bewley, J.D.,The Expression of Dehydrin Proteins in DesiccationSensitive (Recalcitrant) Seeds of Temperate Trees, Planta,1994, vol. 193, pp. 478–485.

27. Farrant, J.M., Pammenter, N.W., Berjak, P., Farn-sworth, E.J., and Vertucci, C.W., Presence of Dehydrin-Like Proteins and Levels of Abscisic Acid in Recalcitrant(Desiccation Sensitive) Seeds May Be Related to Habi-tat, Seed Sci. Res., 1996, vol. 6, pp. 175–182.

proteins of cotyledons of mature horse chestnut seeds

Documents