Plant Physiol. (1969) 44, 1428-1438

The Effect of Ultracentrifugation on Fine Structure and a.AmylaseProduction in Barley Aleurone Cells1

Russell L. JonesUniversity of California, Berkeley, California 94720

Received May 22, 1969.

Abstract. Ultracentrifugation of barley aleurone cells results in the stratifioation oforganelles thus allowing for a quantitation of those organelles. Gibberellic acid (GA8) -stimu-lated a-amylase production in stratified cells is reduced by centrifugation at gravitationalforces greater than 40,000g. Forces below 30,000g do not affect GA3.;stimulated a-amylaseproduotion although stratification of organelles occurs at these forces. The ability of centrifugedoells to respond maximally to GA8 by producing a-amylase is related to the degree of redis-tribution of organelles within these cells. Thus, recovery of cells from centrifugation at foroesbelow 30,000g is rapid, while recovery from forces above 40,000g is slow.

Many workers have now estaiblished that gibberel-lic acid (GA.) enhances the formation of severalhydrolytic enzymes in the aleurone layer of barleyseed (2,11,12,13). Filner and Varner (3) andJacobsen and Varner (5) have shown respectivelythat the de novo synthesis of a-amylase and a pro-tease occurs after the application of GA3.

Recently, attempts have been made to correlatefine structural changes with a-amylase productionin barley aleurone cells following GA. treatment (7).Such ultrastructural changes can be detected inaleurone layers within 2 hr of GA3 application, thechanges becoming more pronounced with duration ofexposure of the cells to GA3 (7). Among the firstfine-structural changes observed are those apparentlyrelated to an increase in the overall volume of thealeurone grains and include an increase in the amountof endoplasmic reticulum (ER). However, the pre-cise quantitation of these and other GA3-stimulatedfine-structural changes is difficult using conventionallimited samplinig of random sections. Loud (10)has described a method of quantitating organelles in2-dimensional electron micrographs but this methodis not suitable for quantitation when large samplesizes must be used.

In order to obtain an estimate of the organellesin barley aleurone cells we have used the organellestratification techniques described for plant cells byBouck (1). Bouck showed that when root tips ofPisum sativum were subjected to ultracentrifugation,the various cell organelles showed a characteristicdistribution within the cell. Sutbsequent aseptic cul-ture of such stratified cells indicated that they had

1 Supported by National Science Foundation grantGB-8332.

not been noticeably affected 'by exposure to highgravitational force.

In addition to obtaining information concerningquantitative changes in the organelles of barleyaleurone cells, stratification should indicate the im-portance of subcellular organization with respect toa given physiological response. Thus, this paperwill attempt to determine whether the synthesis ofhydrolytic enzymes in response to GA3 treatment isa function of the presence of certain cellular com-ponents or whether the organization of these com-ponents in a specific manner is a necessary pre-requisite for this synthesis.

Methods

Barley (Hordeum vulgare L. cv. Himalaya)seeds, 1965 harvest, were used throughout this in-vestigation. Em;bryoless barley half-seeds weresterilized in a 10 % Clorox solution for 30 min.washed with sterile water and allowed to imbibe onsand for 2 days. For centrifugation, aleurone layerswere removed from the starchy endosperm, placedin cellulose nitrate tu3bes sterilized in 10 % Cloroxsolution, and the tubes filled with sterile water. Inall cases, centrifugation was carried out at 20. Thetissue was prepared for microscopy as describedpreviously 1(6). For electron microscopy, aleuronetissue was fixed in either 2 % KMnO4 or in 3 %glutaraldehyde-2 % osmium. The KMnOQ-fixedtissue was embedded in Epon and the sectionswere post-stained in Reynold's lead citrate while theglutaraldehyde-osmium fixed tissue was embedded inEpon and the sections post-stained in saturateduranyl acetate followed by Reynold's lead citrate.For lght microscopy, aleurone layers were fixed in

1428

JONES-ULTRACENTRIFUGATION OF BARLEY ALEURON E LAYERS 1429

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FIG. 1. Electron micrograph of barley aleurone cell imbibed on sand for 2 days. Note the random distribution ofthe aleurone grains (AG), spherosomes (S) and other organelles. KMnO4 fixation. X 9,000. Insert showslight micrograph of aleurone cell imbibed on sand for 2 days. X 1,800.

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JONES-ULTRACENTRIFUGATION OF BARLEY ALEURONE LAYERS

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FIG. 2. Barley aleurone cell following ultracentrifugation for 2 hr at 90 000y. The arrowv indicates the direc-tion of the applied centrifugal force (F). Spherosomes (S) are located at the centripetal pole of the cell while AGare found at the centrifugal pole. Mitochondria (M), dictyosoines (D), endoplasmic reticulum (ER), microbodies(MB) and plastids (P) are found between the AG and S. KMnO4 fixation. X 5850. Insert shows light micro-graph of aleurone cell centrifuged at 90,00(g for 2 hr. Note prominenit stratification of organelles. X 1400.Insert

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FIG. 3. a. Portioni of an aleurone cell centrifuged for 2 hr at 90,000g show,%ing the region between aleuronegrains (AG) and( spiiercsomes (S). 'Note the position of the dictyosomnes (D) relative to the other organielles inthis region. KM.\nO4 fixation. X 17,500. b. GAld-osmiur-n fixation of aleurone cell centrifugcd for 2 hr at 90,000g.The region between AG anid S is show\n. -Note the rough ER (RER) and mnitcclhondria (MI). >X 22,750. Arrowindicates the directioni of the applied centrifugal force (F).

1433

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JONES-ULTRACENTRIFUGATION OF BARLEY ALEURONE LAYERS

3 % glutaraldehyde and embedded in Epon. Sec-tions were cut at 1 to 2 ,A and stained with theAniline blue-black and periodic acid-Schiff's reagents.

ca-Amylase present in the incu;bation medium andtissue extracts was determined colorimetrically by thestarch iodine method and the results were expressedas units of cx-amylase (9). The incorporation of"C leucine and 14C uridine was determined by themethods described by Chrispeels and Varner (2).

Results

Barley aleurone cells are characterized by thepresence of numerous aleurone grains (AG) andspherosomes (Fig. 1) as previously reported (6. 7).Ultracentrifugation of barley aleurone layers at90,000g for 2 hr results in the displacement of theseorganelles. The lipid-containing spherosomes mi-grate centripetally and the protein-containing aleu-rone grains migrate centrifugally (Fig. 2). Thenucleus, endoplasmic reticulum, dictyosomes, mito-chondria, microbodies and leucoplasts become strati-fied between the zones occupied by the spherosornesand the aleuronie grains (Figs. 2 and 3).

The centripetal zone of spherosomes is essentiallyhomogenous although occasionally mitochondria andfragments of ER become trapped among theseorganelles (Fig. 2). Similarly the centrifugal poleof the aleurone cell consists mainly of aleuronegrains (Fig. 2) though occasionally spherosomesremain associated with the aleurone grains. In lightof their markedly different buoyant densities, thepersistant association of the AG and spherosomesfollowing centrifugation indicates that these 2 or-

ganelles may be physically associated in some way.It is of interest to note that the spherosomes remainassociated within the AG during GA3-stimulatedhydrolase synthesis even when most of the storedreserves of the AG have been depleted (8).

The zone between the aleurone grains and sphero-somes is heterogenous (Figs. 2 and 3) but stratifica-tion of the organelles present in this zone occurs.Particularly, the dictyosomes stratify in a regionadjacent to the spherosomes (Fig. 3a).

This stratification of organelles is also visible atthe light microscope level, the zones occupied by thespherosomes and aleurone grains being readily iden-tified at opposite poles of the cells (Figs. 1 and 2).

When aleurone cells which have been stratifiedat 90,000g for 2 hr are subsequently exposed to GA3,a marked inhibition of a-amylase production isobserved relative to uncentrifuged cells (Fig. 4).This inhibition of ac-amylase production is a functionof the gravitational force applied to the aleuronecells. Thus, ultracentrifugation at 20,000g for 24 hrdoes not result in an inhibition of oa-amylase pro-duction, while forces of 40,000g and 60,000g for 24hr result in a 20 and 60 % inhibition of a-amylaseproduction respectively (Fig. 4). Ultracentrifuga-

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Time of centrifugotion (hours)FIG. 4. Graph relating force and time of centrifugation

to Cx-amylase synthesis. Isolated aleurone layers were

centrifuged and subsequently exposed to 0.5 ,ug/ml GA.for 24 hr. a-Amylase measured in the incubation mediumafter 24 hr of incubation with GA3.

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gation at high gravitational forces for 1 hr and GA3-stimulated ca-amylase production. Isolated aleuronelayers exposed to GA. (0.5 ,ug/ml) for 24 hr aftercentrifugation.

tion at 300,000g for 2 hr results in an almost com-

plete inhibition of enzyme synthesis i(Fig. 4). Atlower speeds of centrifugation (30,000g and below)the effect of stratification on a-amylase productionis independent of the duration of centrifugation(Fig. 4), while at speeds exceeding 40,000g, inhibi-tion of a-amylase production is a function of theduration of the centrifugal force (Fig. 4) and thetotal force applied (Fig. 5).

Although exposure of aleurone cells to gravita-tional forces of 20,000g does not result in inhibition

1435

PLANT PHYSIOLOGY

Table II. Oxygen Uptake in Control and CentrifugedA leurone Layers

Aleurone layers and half seeds were centrifuged for2 hr at 90,000g and incubated in buffer in a Warburgflask. Manometric readings were continued for 4 hrafter centrifugation.

Treatment

ControlCentrifuged

2 3

Centrifugal force (g x 105 )FIG. 6. a-Amylase production in aleurone cells ex-

posed to GA3 (0.5 Ag/ml) immediately, 24, 48, and 60hr after ultracentrifugation. a-Amylase measured 24hr after GA 3 treatment.

of a-amylase production. it does result in the strati-fication of the cellular organelles. However, ex-amination of aleurone cells 2 hr after centrifugatioiiat 20,000g indicates considerable redistribution ofthe organelles withini the cell such that the stratifi-cation pattern is lost. Thus the inhibition o-f a-amy-lase production following ultracentrifugation seemsto be a function of the ability of the cells to recoverfrom the applied centrifugal force. When aleuronepieces are centrifuged at 90,000g for 2 hr and ex-posed to GAs immediatelv, a-amylase production isinhibited 65 % relative to the controls. However.if GA3 treatment is delayed for 24 or 48 hr, a-amy-lase production recovers to within 14 and 4 % of thecontrols respectively (Fig. 6). Recovery of theability to produce a-amylase in response to GA?seems directly related to the degree of redistributionof organelles withini the cell.

The a-amylase produced by barley aleurone cellsis released into the incubation medium by an activesecretory mechanism (2, 8). The inhibition ofa-amylase production by centrifugation could there-

Table I. Extracted and Secretd 0-amylase Froml GA3Treated Aleurone Cells Following CentrifugationAleurone layers were centrifuged at 90,000Xg for 2

hr at 20 while control aleurone layers were maintainedat 20 for a 2 hr period. Aleurone layers were thenexposed to 0.5 jug/ml GA3. a-amylase was measured inthe incubation medium and in extracts of the cells 24hir after treatment with GA3.

a-Amylase UnitsTreatment Medium Extract Total

Control 53 12 65Centrifuged 16 12 28

ul 02/hr.Xleurone layers 5 Half seeds

11.3 18.514.7 20.3

Table III. Inicorporation of '4C-leucine Into Trichloro-acetic Acid-precipitable Proteins of Centrifuged

and Control Aleitrone LayersAleurone layers were centrifuged for 2 hr at 90,000g

then exposed to 2 uc of 14C-leucine (250 mC/mM) and0.002 M cold leucine for 3 hr. Aleurone layers extractedwith 0.2 M NaCl and the proteins precipitated from theseextracts with 15 % trichloroacetic acid (TCA). Theprecipitated protein wvas collected on millipore filters andcounted in a gas-flow counter.

Treatment cpm in TCA-precipitable proteins

Control 13 200Centrifuged 13 920

fore be mediated bv ain effect oni enzyvlme secretioniand would result in an accumniulationi of a-amvlasewithin the cell. Extraction of centrifuged aleuronecells does not indicate atn effect of gravitational forceon enzyme secretioni (table I). Likewvise it couldbe argued that inhibitionl of a-aiuvlase production ismediated via ain effect on respiration since Varner(12) has shown that a-amylase production is respira-tion-dependent. Centrifugation did not affect oxvgenuptake as measured by Warburg nmanometry (tableII) although the mitochondria were stratified withinthe cells (Fig. 2 and 3). Similarly, gravitationalforce did not affect the incorporation of 14C-leucineinto trichloracetic acid-precipitable proteins (tableIII) or the incorporation of 14C-uridiine into salt-soluble RNA (table IV).

Table IV. Incorporationt of A4C-uridine Into SaltSoluble RNA, in Centrifuged and Control

Aleurone CellsAleurone cells were centrifuged at 90,000y for 2

hr and then exposed to 1 AC of 14C-uridine (25 mC/mM)for 4 hr. Aleurone layers were extracted with 1 M NaClcontaining 1.25 mg/ml RNA. The RNA was precipitatedwith 15 % trichloroacetic acid, collected on a milliporefilter and counted in a gas-flow counter.

Treatment cpm in salt soluble RNA

Control 980Centrifuged 770

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JONES-ULTRACENTRIFUGATION OF BARLEY ALEURONE LAYERS

Discussion

Ultracentrifugation of barley aleurone cells resultsin the stratificationi of organelles within these cells.This stratification is facilitated by the presence inaleurone cells of organelles of markedly differentbuovant densities. Thus, the lipid-containing sphero-somes migrate centripetally while the more densealeurone grains migrate centrifugally within the cell.The zonation allows for the rough quantitation ofthe organelles within the cell. Since the sphero-somes and aleurone grains possess densities whichare markedly different from each other as well asfrom the other organelles, the zones which theyoccupy within the cells can be readily measured byplanimetry or weighing techniques. Likewise thismethod of quantitation can be applied to the organelleswhich migrate to the region between the spherosomesand aleurone grains. Quantitation of the endoplasmicreticulum (ER) on electron micrographs is generallyextremely difficult. After centrifugation however,the amount of ER can be readily determined andchanges in ER with a given treatment (e.g. GA3)can thus be followed.

In addition to offering a means of quantitatingchanges in cell organelles, stratification can serve topartially characterize organelles on the basis ofbuoyant density. This feature would appear to beideally suited for a histochemical study of cellorganelles. For example, aleurone cells containmicrobodies (Fig. 3a) whose contents are thought toinclude enzymes characteristic of lysosomal particles.Ilowever, because of their size '(1-2 u) their de-tection as isolated organelles by histochemical meth-ods at the light microscope level has proven difficult.If the cells containing microbodies are stratified.however, the association of several microbodies in acharacteristic position in the cell should allow for apossible identification of these organelles. I haveused this feature of the stratification method toestablish the lipid nature of spherosomes. Thespherosomes of uncentrifuged cells cannot be recog-nized as lipid containing organelles using conven-tional histochemical methods. However, if stratifiedcells are examined histochemically for lipoidal mate-rials, the centripetal zone of spherosomes is readilyrecognize(d (Figs. 1 and 2).

Organelle stratification by gravitational force canserve to distingtlish between organelles of similarmorphology hut differing buoyant densities. Anexaniple is given by the difficulty experienced byseveral workers in distinguishing between sphero-somes and microbodies on the basis of their mor-phology (4). However, since spherosomes containlipids and microbodies contain proteins, they canbe identified on the basis of their different buoyantdensities in centrifuged cells (Fig. 3a).

It must be pointed out, that in order to obtainsatisfactory quantitation of organelles from electronmicrographs of stratified cells, the plane of section

must parallel the plane of the applied centrifugalforce. Thus, orientation of tissue during centrifu-gation and sectioning must be carefullv controlled.

Also, the region of the cell from whlich sectionsare taken must be controlled. The nucleus couldfunction as a marker organelle, that is, only thosesections containing a nucleus could be used forquantitation or vice versa. In aleurone cells, sectionswithout a nucleus are generally taken since thenucleus occupies much of the zone between sphero-somes and aleurone grains, thus displacing some ofthe ER, mitochondria, dictyosomes and leucoplastsfrom that plane.

Stratification of barley aleurone cells at forcesgreater than 40,000g results in a significant reductionin GA3-stimulated a-amylase production (Figs. 4and 5). Gravitational forces below 20,000g do notcause a reduction in a-amylase formation (Fig. 4).However, examination of cells centrifuged at thisforce indicated that stratification of organelles hadoccurred although a-amylase production was un-affected. The recovery of these cells from the strati-fication procedure is rapid, complete redistributionof the organelles occurring within 4 hr of centrifuga-tion. Cells which have been centr-ifuged at forcesof 40,000g and greater do not recover as rapidly.Redistribution of organelles, and recovery of theability to produce a-amylase maxinmally in aleuronecells centrifuged for 2 hr at 90,000g, does not occuruntil approximately 48 hr after centrifugation (Fig.6). Thus, the response of stratified cells to GA,as measured by a-amylase l)roduction is a functionof the rate of redistribution of the organelles.

The effect of stratification of aleurone cells onsubsequent a-amvlase productionl does not appear tobe nmediated via an effect on respiration or RNA andprotein synthesis. Oxygen uptake by barley aleuronecells as measured by Warburg manometry was notaffected by stratification (table II) ; neither was theabilitv of these cells to incorporate 14C-leucine intotrichloracetic acid-precipitable protein (table III) or14C-uridine into salt-soluble RNA (table IV). Inaddition, Ferrari (personal communication) hasshown that nitrate reductase induction by nitrate isnot impaired by stratification.

It is suggested therefore, that in barley aleuronecells, the ability to respond to GA, is a function notonly of the presence of distinct cellular componentsbut also of the distributioni of these componentsrelative to each other within these cells. Thus,when the cell organelles of barley are subjected tophysical separation, the ability to make large amountsof hydrolytic enzymes is impaired although somephysiological functions are not.

Acknowledgment

The skillful technical assistance of Mrs. Janet Priceis gratefully acknowledged.

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PLANT PHYSIOLOGY

Literature Cited

1. BOUCK, G. B. 1963. Stratification and subsequentbehavior of plant cell organelles. J. Cell Biol.18: 441-57.

2. CHRISPEELS, M. J. AND J. E. VARNER. 1967. Gib-berellic acid-induced synthesis and release of a-

amylase and ribonuclease from barley aleuronelayers. Plant Physiol. 42: 398406.

3. FILNER, P. AND J. E. VARNER. 1967. A simple andunequivocal test for de novo synthesis of enzymes:density labeling of barley a-amylase with H2180.Proc. Natl. Acad. Sci. Wash. 58: 1520-26.

4. FREDERICK, S. E., E. H. NEWCOMB, E. L. VIGIL, ANDW. P. WERGIN. 1968. Fine structure character-ization of plant microbodies. Planta 81: 229-52.

5. JACOBSEN, J. V. AND J. E. VARNER. 1967. Gibber-ellic acid induced synthesis of protease by isolatedaleurone layers of barley. Plant Physiol. 42: 1596-1600.

6. JONES, R. L. 1969. The fine structure of barleyaleurone layers. Planta 85: 359-75.

7. JoNES, R. L. 1969. Gibberellic acid and the finestructure of barley aleurone cells. I. Changesduring the lag phase of a-amylase synthesis. Planta87: 119-33.

8. JONEs, R. L. 1969. Gibberellic acid and the finestructure of barley aleurone cells. II. Changesduring the synthesis and secretion of a-amylase.Planta 88: 73-86.

9. JONES, R. L. AND J. E. VARNER. 1967. The bio-assay of gibberellins. Planta 72: 53-59.

10. LouD, A. V. 1962. Method for the quantitativeestimation of cytoplasmic structures. J. Cell Biol.15: 481-87.

11. PALEG, L. 1960. Physiological effects of gibberellicacid. II. On starch hydrolyzing enzymes of bar-ley endosperm. Plant Physiol. 35: 902-06.

12. VARNER, J. E. 1964. Gibberellic acid controlledsynthesis of a-amylase in barley endosperm. PlantPhysiol. 39: 413-15.

13. YoMo, H. 1960. Studies on the a-amylase activat-ing substance. IV. On the amylase activatingaction of gibberellin. Hakko Kyokaishi 18: 600-02.

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