Plant Physiol. (1983) 73, 746-7530032-0889/83/73/0746/08/$00.50/0

Fractionation of Nucleoli from Auxin-Treated SoybeanHypocotyl into Nucleolar Chromatin and Preribosomal Particles'

Received for publication April 11, 1983 and in revised form July 12, 1983

YIE-MING CHEN, DER-HWA HUANG2, SHAW-FANG LIN, CHU-YUNG LIN3, AND JOE L. KEYDepartment ofBotany, National Taiwan University, Taipei, Taiwan (Y. M. C., S. F. L., C. Y. L.); andDepartment ofBotany, The University ofGeorgia, Athens, Georgia, 30602 (D. H. H., J. L. K.)

ABSTRACr

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne orKaoshiung No. 3) were isolated and purified by Percoll density gradientcentrifugation. There was a 2.1-fold increase in RNA and a 2.8foldincrease in protein after a 24-h auxin treatment per unit nucleolar DNA.More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.

Nucleoli from auxin-treated tissue were fractionated by suspension in20 millimolar dithiothreitol at room temperature for 20 minutes into twodistinct fractions referred to as the nucleolar chromatin and preribosomalparticle fractions. The DNA:RNA:protein ratio ofthe chromatin fractionwas 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNArecovered in this fraction. The acid-soluble proteins in the chromatinwere resolved into 32 protein spots on 2-D gel electropherogram. Themost abundant spots were identified as histones.The nucleolar preribosomal particle fraction had a DNA:RNA:protein

ratio of 1:24:102 and contained only trace amounts of RNA polymerase1 activity and only 10 per cent of the nucleolar DNA. Acid-solubleproteins associated with these particles were resolved into 78 proteinspots; 72 of these (acid-soluble) protein spots corresponded in 2-D gelelectrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 proteinspots found in 80S ribosomal proteins were absent in the preribosomalparticles. It seems reasonable, based on these data, that the enlargementof nucleoli after auxin treatment is primarily due to the large increase inribosomal proteins and rRNA which accumulate and assemble in thenucleoli in the form of preribosomal particles.

A substantial body of literature has established that the syn-thesis and processing of pre-rRNA occurs in the nucleolus ofeukaryotic organisms (2, 22, 29, 31). The nucleolar organizerregion (15) contains several hundred to several thousand copiesofrRNA genes which are associated with proteins in the form ofnucleolar chromatin. During the transcription of pre-rRNA,several ribosomal proteins become associated with the nascentRNA; this leads to further processing and packaging of the pre-rRNA in the nucleolus to form ribonucleoprotein complexestermed preribosomal particles or preribosomes (29).The nucleolar volume as a fraction of total nuclear volume

varies considerably as tissues undergo altered rates of ribosomeproduction. For example, partial hepatectomy or injection ofthioacetamide into rats results in an increase in relative nucleolar

'Supported by National Science Council of Taiwan under GrantNSC70-0201-B02-01 to C. Y. L. and Y. M. C. and National Institute ofHealth Grant 5ROIGM30317-03 to J. L. K.

2 Present Address: Box 237, Rockefeller University, New York, NY10021.3To whom correspondence should be addressed.

volume to as much as 75 per cent of nuclear volume (1, 4) ofliver tissue; this increase in nucleolar volume correlates withenhanced rRNA synthesis and accumulation of ribonucleopro-tein or preribosome particles in the nucleolus. Auxin treatmentof quiescent mature hypocotyl tissue of soybean leads to adramatic increase in nucleolar volume in association with an 8-to 10-fold increase in the rate ofrRNA synthesis (7, 10, 19). Thevery large increase in ribosome accumulation following auxintreatment makes the soybean system an attractive model forassessing the relationship between rRNA synthesis and the syn-thesis of ribosomal proteins (9, 30) as well as for an analysis ofthe mechanism(s) operative in the modulation ofRNA polymer-ase I activity and nucleolar function (20). In an earlier paper, wereported on the isolation of nucleoli from auxin-treated soybeanhypocotyls and the association ofRNA polymerase I with thosenucleoli (19). In order to begin a detailed analysis of nucleolarproteins relative to the auxin-induced increase in rRNA synthesisand associated increase in nucleolar volume, we report here onan improved procedure for nucleolar isolation and the resolutionof those nucleoli into preribosome particles and nucleolar chro-matin. Some broad comparisons are made between nucleoli ofcontrol and auxin-treated tissue.

MATERIALS AND METHODS

Plant Materials. Soybean seeds (Glycine max L. var Wayneor Kaoshiung No. 3) were germinated in moist vermiculite inthe dark at 28C. Three-d-old seedlings were sprayed with 2.5mM 2,4-D (pH 6.0), and the hypocotyles were harvested 24 hafter spraying.

Chemicals. Bistris4, Mes, DTT, 2-MSH, calf thymus DNA,ATP, GTP, CTP, UTP, TEMED, thioglycolic acid, sucrose, andPercoll were purchased from Sigma. The a-amanitin was pur-chased from Calbiochem. Acrylamide, MBA, and SDS werepurchased from Merck. All other organic chemicals were reagentgrade.

Preparation of Nucleoli. The procedure for preparation ofnucleoli was based on the method of Lin et al. (19) with somemodifications. Eighty g of precooled tissue were mixed with 80ml of grinding buffer containing 25 mM Mes-NaOH (pH 6.0),10 mM KCI, 10 mM MgCl2, 30 per cent glycerol (v/v), 10 mM 2-MSH, and 0.6 M sucrose (buffer A), and homogenized with theWillems polytron (Brinkman, PS-20-St) for 2 min at low setting.The tissue homogenate was filtered through four layers of cheesecloth and then two layers of Miracloth. Triton X-100 was addedto the filtrate to a final concentration of 1 per cent (v/v) followedby gentle stirring with the Willems polytron, and then centrifu-gation for 15 min at 6,000g. The pellet was resuspended in 25

4Abbreviations: Bistris, bis-(2-hydroxyethyl)-imino-tris(hydroxy-methyl)methane; 2-MSH, 2-mercaptoethanol; TEMED, N,N,N',N'-tetramethylenediamine; MBA, methylene bis-acrylamide; 2-D, two-di-mensional.

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NUCLEOLAR CHROMATIN AND PRERIBOSOMAL PARTICLES

±

S_

A4_.

C,)

a'

*...

:_.

MW

_- - 94

Am - 68

-43

_ 2-30

-21^.l

OW. - 14.3

FIG. 1. Two-dimensional gel electrophoresis of acid-soluble proteins from auxin-treated nucleoli. The mol wt standards from the top to thebottom are 94 kD, 68 kD, 43 kD, 30 kD, 21 kD, and 14.3 kD, respectively.

mM Mes buffer (pH 6.0) containing 20 mM KCI, 5 mm MgCl2,1.2 M sucrose, and 10 mm 2-MSH (buffer B). The crude nucleolarsuspension was loaded onto Percoll gradients prepared by layer-ing 7 ml each of 40 per cent and 60 per cent Percoll in buffer B.The gradients were centrifuged in a Sorvall HB-4 rotor for 30min at 10,000g. The interface between the 40 per cent and 60per cent Percoll layers was removed, mixed with an equal volumeof buffer B, and centrifuged at 8,000g for 10 min. This pelletwas washed with buffer B by repeated suspension and centrifu-gation. The Percoll gradients removed starch and other lightercontaminants and proved useful for the large scale isolation ofhighly purified nucleoli from plant tissues.

Preparation of Nucleolar Chromatin and Preribosomal Parti-cles. The purified nucleoli were suspended with a small volumeof 10 mm Tris-HCl buffer (pH 8.0), 20 mm KCI, 1 mM MgCl2,250 mm sucrose, and 20 mm DTT (buffer C), and gently stirredat room temperature for 20 min (28). The pellet and the super-natant obtained after centrifugation at 20,000g for 30 min weredesignated as 'nucleolar chromatin' fraction and 'preribosomalparticle' fraction, respectively. The supernatant preribosomalparticle fraction was subjected to one ofthe following treatments:(a) precipitation by the addition of 6 volumes of cold acetone,for determination of DNA, RNA, and protein contents; or (b)centrifugation at 200,000g for 90 min to pellet the preribosomal

particles by layering the supernatant fraction on a cushion of 1.5M sucrose in 50 mM Tris-HCI buffer (pH 8.5) containing 20 mMKCI and 5 mM MgCl2 (buffer D).

Preparation of Ribosomes. Monomeric ribosomes were pre-pared from the root tips of 60-h germinated etiolated soybeanseedlings which were immersed in water and bubbled with N2gas at room temperature for 2 h (18). Sections of 0.5-cm roottips were harvested and chilled. About 1 g of tissue was homog-enized in 15 ml of 250 mm sucrose in buffer D with the Willemspolytron at high speed for 10 s. The homogenate was filteredthrough two layers ofcheese cloth and Miracloth, and centrifugedat 20,000g for 30 min. The supernatant was layered over 5 mlofbuffer D containing 1.5 M sucrose, and centrifuged at 200,000gfor 90 min in a MSE type 64 rotor. The ribosomal pellet wassuspended in buffer D for extraction of ribosomal proteins.

Extraction of Basic Proteins. The method of Hardy et al. ( 12)for the extraction of basic proteins was used. To one volume ofnucleoli, nucleolar chromatin, preribosomal particles or ribo-somes in buffer D, one-tenth volume of 1 M MgCl2 was added,followed by drop-wise addition of two volumes of glacial aceticacid. The mixture was stirred for 45 min, and centrifuged for 30min at 20,000g. After centrifugation, the supernatant was mixedwith six volumes of precooled acetone (below 0°C), and themixture was held at -20°C for at least 3 h. The precipitate was

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Plant Physiol. Vol. 73, 1983

±

U)

C')

FIG. 2. Two-dimensional gel electrophoresis ofacid-soluble proteins from nucleolar chromatin obtained by fractionation ofauxin-treated nucleoliperformed according to the procedures outlined in "Materials and Methods." Arrows indicate histones.

Table I. RNA Polymerase I and II Activities in Isolated Nucleoli andin the Nucleolar Chromatin and Preribosomal Particle FractionsRNA polymerase activity was assayed by the method of Chen et al.

(7).RNA Polymerase Activity

FractionsI II

pmol/lMg DNANucleoli (initial)a 8.24 0.72Nucleoli (final)b 6.84 0.60Nucleolar chromatin 5.77 0.70Preribosomal particles 0.82 2.07

a RNA polymerase activity was obtained from Percoll purified nucleolibefore suspension in the nucleoli fractionation medium.

b RNA polymerase activity was obtained 20 min after suspension inthe nucleoli fractionation medium with gentle stirring at room temper-ature.

collected by centrifugation at 20,000g for 10 min, dried in vacuo,and dissolved in 8 M urea containing 10 mm DTT for at least 10h at room temperature, and then stored at -20C.

Histone Extraction. Histones were obtained by H2S04 extrac-tion of purified nuclei from control soybean hypocotyl as de-scribed by Spiker et al. (27).

Table II. Relative Contents in DNA, RNA, and Proteins in IsolatedNucleoli, Nucleolar Chromatin, and Preribosomal Particles

Fractions DNA RNA Proteins

MgNucleoli' 100 470 2,300Nucleolar chromatinb 90 230 1,280Preribosomal particles 10 240 1,020

a Data are based on a nucleolar fraction containing 100 Mg DNA.b Data on the two subtractions are calculated to represent those from

an isolated nucleolar fraction containing 100 gg DNA assuming a 100%recovery. The recovery of each fraction was about 90 to 95%.

Determination of DNA, RNA, and Protein in Nucleoli, Nu-cleolar Chromatin, and Preribosomal Particle Fraction. The iso-lation ofDNA and RNA from each fraction followed the methodof Howell (13). RNA content was determined by the orcinolmethod (26), using yeast RNA as a standard. DNA content wasdetermined by the diphenylamine method (3), using calfthymusDNA as a standard. Protein was determined by the method ofLowry (21), using BSA as a standard.Assay for In Vitro Synthesis. RNA polymerase activity was

assayed by the method of Chen et al. (7). The 0.25-ml reactionmixture contained 50 mm Tris-HCl (pH 8.0), 10 mm DTT, 5mM MgCl2, 29 per cent glycerol, 0.4 mm each of ATP, GTP,

748 CHEN ET AL.

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NUCLEOLAR CHROMATIN AND PRERIBOSOMAL PARTICLES

g-l1l-------.

!~~~~~~~~~~~N-:QX~~~~~~~~~~~m

FIG. 3. Two-dimensional gel electrophoresis of histones, from control nuclei.

CTP, and 0.02 mM UTP containing 1 ACi of [3H]UTP. Thereaction was at 280C for 20 min after the addition of nucleoli,nucleolar chromatin, or preribosomal particle fraction, and wasterminated by the addition of 2 ml of cold 10 per cent TCA. Theacid-precipitable material was collected on Whatman GF/C glassfilters, and washed with cold 5 per cent TCA and 95 per centethanol. The filters were dried, and radioactivity was measuredin a Beckman LS- I00 liquid scintillation spectrometer.Two-Dimensional Gel Electrophoresis of Acid-Soluble Pro-

teins. The technique developed by Mets and Bogorad (24) wasemployed with some modifications for fractionation of acid-extracted proteins. For the first dimension, a gel mixture con-taining 4 per cent acrylamide, 0.1 per cent MBA, 8 M urea, 0.057M Bistris (pH 6.0) was prepared (the pH of Bistris solutions wereadjusted with acetic acid except where otherwise specified). Gelformation was initiated by the addition of 1 Al of TEMED, and5 ,ul of 10 per cent ammonium persulfate were added per ml ofgel mixture. The mixture was poured into 3- X 130-mm glasstubes to a final height of 120 mm. Gels were carefully overlayeredwith 40 Ml of a solution containing 0.057 M Bistris (pH 5.0), and6 M urea, and allowed to stand for 1 h before use. Protein samplessuspended in the urea-DTT solution were vortexed vigorouslyfor at least 5 min and then loaded onto the gel. Upper and lower

reservoir buffers were 0.01 M Bistris (pH 4.0) and 0.179 Mpotassium acetate (titrated to pH 5.0 with acetic acid), respec-tively. An aliquot of basic fuchsin was run in a blank tube as amarker. Proteins were run toward the cathode with a current of1.5 mamp/gel for 5.5 h. The second dimension was run imme-diately after the first electrophoresis. A 30-ml gel mixture con-taining 12.5 per cent acrylamide (acrylamide:MBA = 29.2:0.8and 0. 143 M Bistris-HCI (pH 6.75) was mixed with 15 ,l TEMEDand 150 ,l of 10 per cent ammonium persulfate, and cast in theslab gel apparatus. The gel from the first dimension was fixed onthe top of the slab gel by adding melted 1 per cent (w/v) agarosesolution containing 0.057 M Bistris (pH 5.0). The upper reservoircontained 0.07 M Bistris-Mes (pH 6.5), and the lower 0.07 MBistris-HCl buffer (pH 6.75). Electrophoresis was at 15 mamp/slab for 7 h, and the gel was usually cooled by circulating tapwater. After electrophoresis, the slab gels were removed, fixed ina mixture of 50 per cent isopropanol and 10 per cent TCA for30 min, stained overnight with 0.1 per cent Coomassie blue in25 per cent isopropanol and 10 per cent TCA, and then withseveral changes of 10 per cent ethylacetate, 5 per cent acetic acid,and 7 per cent ethanol. After soaking in a solution containing10 per cent acetic acid and 1 per cent glycerol for 30 min, gelswere dried in a Bio-Rad gel dryer (model 224).

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Plant Physiol. Vol. 73, 1983

+D

.*..

C,)

C,)

FIG. 4. Two-dimensional gel electrophoresis of acid-soluble proteins from preribosomal particles obtained by fractionation of auxin-treatednucleoli according to the procedures outlined in "Materials and Methods." Arrows indicate nonribosomal proteins.

One-Dimensional SDS Gel Electrophoresis. The acid-insolu-ble residue of nucleolar chromatin was dissolved in Laemmli'ssample buffer (10 per cent glycerol, 5 per cent 2-MSH and 0.0625M Tris [pH 6.8], and 2.3 per cent SDS), boiled for 1 min. One-dimensional SDS gel electrophoresis was performed according toLaemmli (16).

RESULTSRNA Polymerase Activities in Isolated Nucleoli, Nucleolar

Chromatin, and Preribosomal Particle Fraction. RNA polymer-ase activity was assayed in the presence and absence of 2 ,Rg/mla-amanitin to distinguish between RNA polymerase I and RNApolymerase II activities (5). RNA polymerase activity measuredin the presence of a-amanitin reflects the activity ofRNA poly-merase I. More than 90 per cent ofRNA polymerase activity inthe nucleolar fraction was due to RNA polymerase I, and thiswas restricted essentially to the nucleolar chromatin fraction.The preribosomal particle fraction contained only a small frac-tion ofthe total RNA polymerase activity and this was primarilyRNA polymerase II activity (Table I). Thus, the nucleolar frac-tion and its subtractions were largely free from contaminationby extra-nucleolar chromatin.

The DNA:RNA:protein ratio of nucleoli from auxin-treatedtissue was 1:4.7:23. The nucleolar preribosomal particle fractionhad a DNA:RNA:protein ratio of 1:24:102 and contained only10 per cent of the total DNA of nucleoli. This small amount ofDNA might be due to contamination by nucleolar chromatinfragments or to nucleoplasmic chromatin (Table I). The nucleo-lar chromatin fraction contained a DNA:RNA:protein ratio of1:2.5:14 and about 90 per cent of the nucleolar DNA (Table II).These values for the distribution of macromolecules and RNApolymerase activities establish separate and distinguishable ident-ities for the two fractions.Two-Dimensional Gel Electrophoresis of Acid-Soluble Pro-

teins from Nucleoli, Nucleolar Chromatin, Preribosomal Parti-cles, and 80S Cytoplasmic Ribosomes. Acid-soluble proteinsfrom the auxin-treated nucleoli were resolved into approximately170 protein spots in 2-D gels run according to the methods ofMets and Bogorad (24) (Fig. 1) and about 32 protein spots wereresolved from the nucleolar chromatin (Fig. 2). To ascertainwhich of the nucleolar chromatin proteins correspond to his-tones, a comparative analysis of these proteins with histonesextracted from untreated soybean hypocotyl nuclei was made.The 2-D gel pattern of soybean histones is displayed in Figure 3.

750 CHEN ET AL.

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NUCLEOLAR CHROMATIN AND PRERIBOSOMAL PARTICLES

l:j

.L.

±

.

C)

(I).4.

FIG. 5. Two-dimensional gel electrophoresis of 80S cytoplasmic ribosomal proteins. Electrophoretic separations were performed according to theprocedures outlined in "Materials and Methods." Arrows indicate mature 80S ribosomal proteins not found in preribosomal particles.

Table III. Comparison ofMacromolecular Composition in Isolated Nucleolifrom Control and Auxin-TreatedSoybean Hypocotyl

The data are normalized to the basis of I ;sg DNA.

Acid-Soluble Acid-Insoluble RNA RNAPlant Tissue DNA RNA Protein Protein Polymerase I Polymerase IIProtein ~~~~Activitya Activitya

Ag pmol/lg DNAControl 1.00 2.29 7.60 0.93 1.0 0.67Auxin-treated 1.00 4.70 21.20 1.80 8.84 0.72a RNA polymerase I and II activities were obtained as described previously (7).

The comparison indicates that the most abundant protein spotsin nucleolar chromatin coincided with nuclear histones (fromcontrol tissues) (arrows in Fig. 2). More than 80 per cent of theacid-soluble proteins associated with the nucleolar chromatinfraction appear to be histones based on intensity of staining.About 78 protein spots were seen in 2-D electropherograms of

the preribosomal particle fraction (Fig. 4). Those acid-solubleproteins were compared with 80S monomeric ribosomal proteinsisolated from the root tips of soybean seedlings which wereresolved into 87 protein spots (Fig. 5). Of the 78 protein spots in

preribosomal particles, 72 coincide with spots in the 80S ribo-somal protein gel. Some 15 of the 87 protein spots seen on the80S ribosomal protein gel (indicated by arrows in Fig. 5) wereabsent or substantially reduced in concentration in the preribo-somal particles while there were a few proteins (shown by arrowsin Fig. 4) associated with the preribosome fraction which wasabsent in the mature ribosome.Comparison of Nucleolar Components in Control and Auxin-

Treated Nucleoli. Since rRNA genes are not amplified followingauxin treatment (14), the DNA content per nucleolus from

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Plant Physiol. Vol. 73, 1983

band of nol

Interest wft(I{X

4

- i

-.)-rc 68

- 413

- ~ ~

- j--30-c.

-'1 21

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I 2 3

FIG. 6. One-dimensional SDS gel electrophoresis of acidic proteins from SDS-extracted, acid-insoluble residue of nucleoli. From left to right: (1),mol wt standards; (2), auxin-treated nucleoli; (3), control nucleoli. Equal amount of proteins was loaded in lanes 2 and 3.

control and auxin-treated tissue should be the same. Thus,comparison of content of nucleolar components on the basis of1 ,ug of DNA reflects the relative level of these components incontrol and auxin-treated nucleoli. Chemical compositions ofmacromolecules present in nucleoli from control and auxin-treated tissues are shown in Table III. There was a 2-fold increasein RNA content and almost a 3-fold increase in protein afterauxin treatment. Additionally, RNA polymerase I activity wasenhanced about 8-fold as reported previously (10). The largeincrease in relative protein and RNA content is apparently theresult of a large increase in preribosomal particles and a smallincrease in nucleolar chromatin proteins correlated with theenhancement ofrRNA synthesis and the morphological enlarge-ment of nucleoli.

In addition to the increase in the acid-insoluble proteins ex-tracted from total nucleoli following auxin treatment (Table III),there are some major qualitative differences in the SDS gelprofiles of these acidic proteins isolated from control and auxinnucleoli. As shown in Figure 6, polypeptides with mol wt of59,000 and 52,000 D (bands a and b) decreased in intensity in

auxin-treated nucleoli. In contrast, an increase in intensity afterauxin-treatment was observed for four polypeptides with mol wtof 35,000, 28,500, 27,000, and 18,000 D (bands c, d, e, and f).Generally, it seemed that there was an increase in proteins oflow mol wt in treated nucleoli (lane 2), while in control nucleolisome proteins remained at the top of the gel (lane 3).

DISCUSSIONThe Percoll gradient procedure used here allows the purifica-

tion of morphologically intact nucleoli substantially free of cys-toplasmic and nucleoplasmic contamination based on a numberof criteria. These nucleoli were subsequently fractionated intotwo distinct components, namely nucleolar chromatin and pre-ribosomal particles. The distribution of nucleolar DNA, RNApolymerase I activity, total RNA, total protein as well as quali-tative distribution of basic chromatin and ribosomal proteinsbetween the two fractions establishes the general efficacy of theprocedures used. The ability to fractionate nucleoli as outlinedhere should permit a detailed analysis of nucleolar chromatin formodifications which may relate to the regulation of rRNA syn-

752 CHEN ET AL.

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NUCLEOLAR CHROMATIN AND PRERIBOSOMAL PARTICLES

thesis during normal developmental transitions and auxin-in-duced increases in rRNA synthesis and ribosome formation (20).The large increase in nucleolar volume following auxin treat-

ment seems to relate directly to the large accumulation of preri-bosomal particles composed of rRNA and ribosomal proteinswithin this structure; the relatively small change in the organi-zation/composition ofthe chromatin may contribute to a limitedextent to the enlarged nucleolus. The large increase in volumeseems not to relate to any amplification of the rRNA genesduring the normal or auxin-induced growth transitions based onearlier analyses (14). The rDNA of soybean seems to remain atabout 2,000 copies per nucleolus during auxin treatment.The data presented above show both quantitative and quali-

tative changes in nucleolar chromatin proteins during auxin-mediated changes in rRNA synthesis. Detailed analyses of theseproteins, including synthesis and possible modification duringauxin treatment, should contribute to our understanding of theauxin regulation of rRNA synthesis.The major acid-soluble proteins present in nucleolar chroma-

tin and preribosomal particle fractions are different. In nucleolarchromatin, the major acid-soluble proteins were histones, whilemore than 90 per cent of the acid-soluble protein spots associatedwith preribosomal particles appear to be ribosomal proteins by2-D gel analysis.

Based on the stain intensity of acid-soluble protein spots on 2-D gels, the relative concentration in each spot was differentbetween the preribosomal particle proteins and the 80S cytoplas-mic ribosomal proteins. During the maturation of preribosomalparticles to 80S cytoplasmic ribosomes, the relative amount ofribosomal proteins may change. In preribosomal particles, twosubclasses of acid-soluble proteins differing in biological functionand kinetics of labeling have been reported (30). One subclass iscomposed of ribosomal proteins which are rapidly synthesizedand are transported out of the nucleus. The other subclass isthought to function in the processing of preribosomal particles.Proteins of the latter subclass turn over very slowly and remainin the nucleolus during most of the cell cycle. Under normalcircumstances, a constant amount of preribosomal particles ismaintained in the nucleolus due to a dynamic balance betweenthe synthesis of new preribosomal particles and transport ofprocessed preribosomal particles out of the nucleoli. Therefore,the nucleolus maintains a constant size. As soon as this dynamicbalance is lost, a change of nucleolar size occurs.These studies provide a base of knowledge essential to the

pursuit of the mechanism(s) by which auxin enhances rRNAsynthesis by as much as 8- to 10-fold on a per unit DNA or 15-to 20-fold on a per unit of tissue basis (e.g. soybean hypocotyl)(11). Certainly, a significant proportion of this increased synthesisof rRNA in response to auxin relates to increased levels of RNApolymerase I enzyme and to increased activity of those RNApolymerase I molecules (1 1). Since mature rRNAs (1 8S and 25Sor 0.7 and 1.3 x 106 D, respectively) are initially transcribed asa large precursor molecule of about 2.8 x 105 D in some plants(8, 17, 25) and about 2.3 x 106 in others (6, 28) includingsoybean (15), the rate of processing of these large transcriptscould be involved in establishing the rate of accumulation ofnew ribosomes. Melanson and Ingle (23) in fact have reportedthat precursor processing, including a large increase in the rateof processing of the 1.4 x 106 precursor into the mature 1.3 x106 (25S) rRNA, accounts for a part of the auxin-enhancedsynthesis and accumulaton of ribosomes in artichoke tissue. Theavailability of ribosomal proteins may also play a role in the rateof processing of pre-rRNA and/or accumulation of ribosomes.The level of in vitro translatable ribosomal protein mRNAs hasrecently been shown to increase by some 8-fold following auxintreatment of soybean hypocotyl (9), a value similar to the en-hancement of RNA polymerase I activity following auxin treat-

ment. These data taken together indicate that an understandingof factors involved in enhanced transcription of rRNA andribosomal protein genes as well as posttranscriptional processingevents will be essential if we are to understand normal develop-mental and hormone-regulated modulations in the rate of ribo-some formation. This auxin regulated system should serve as anexcellent model for such studies.

LITERATURE CITEID1. BALLAL NR, H BUSCH 1974 The dynamic state of liver nucleolar proteins as

reflected by their changes during administration of thioacetamide. Life Sci14:1835-1845

2. BROWN DD, JB GURDON 1964 Absence of ribosomal RNA synthesis in theanucleolate mutant ofXenopus laevis. Proc Natl Acad Sci USA 51: 139-146

3. BURTON K 1968 Determination of DNA concentration with diphenylamine.Methods Enzymol 122B: 163-166

4. BUSCH H, K SMETANA 1970 The Nucleolus. Academic Press, New York5. CHAMBON P 1975 Eukaryotic nuclear RNA polymerase. Annu Rev Biochem

44: 613-6386. CHEN D, G SCHULTZ, E KATCHALSKI 1971 Early ribosomal RNA transcription

during germination of wheat embryo. Nature (New Biol) 231: 69-727. CHEN YM, CY LIN, H CHANG, TJ GUILFOYLE, JL KEY 1975 Isolation and

properties of nuclei from control and auxin-treated soybean hypocotyl. PlantPhysiol 56: 78-82

8. Cox BJ, G TURNOCK 1973 Synthesis and processing of ribosomal RNA incultured plant cells. Eur J Biochem 37: 367-376

9. GANTT JS, JL KEY 1983 Auxin-induced changes in the level of translatableribosomal protein messenger ribonucleic acids in soybean hypocotyls. Bio-chemistry. In press

10. GUILFOYLE TJ, CY LIN, YM CHEN, RT NAGAO, JL KEY 1975 Enhancementof soybean RNA polymerase I by auxin. Proc Natl Acad Sci USA 72: 69-72

11. GUILFOYLE T, N OLSZEWSKI, L ZURFLUH 1980 RNA polymerases and tran-scription during developmental transitions in soybean. In CJ Leaver, ed,Genome Organization and Expression in Plants. Plenum Press, New York,pp 93-103

12. HARDY SJS, CG KURLAND, P VOYNOW, G MORA 1969 The ribosomal proteinsof E. coli. 1. Purification of 30S ribosomal proteins. Biochemistry 8: 2897-2905

13. HOWELL SH 1973 The isolation and analysis of DNA from eukaryotic cells.In MJ Chrispells, ed, Molecular Techniques and Approaches in Develop-mental Biology. John Wiley and Sons, New York, pp 117-139

14. INGLE J, J SINCLAIR 1972 Ribosomal RNA genes and plant development.Nature (Lond) 235: 30-32

15. KEY JL 1976 Nucleic acid metabolism. In J Bonner, E Varner, eds, PlantBiochemistry. Academic Press, New York, pp 463-500

16. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature (Lond) 277: 680-685

17. LEAVER CJ, JL KEY 1970 Ribosomal RNA synthesis in plants. J Mol Biol 49:671-680

18. LIN CY, JL KEY 1971 Dissociation of N2 gas-induced monomeric ribosomes:functioning of the derived subunits in protein synthesis in pea. Plant Physiol48: 547-552

19. LIN CY, TJ GUILFOYLE, YM CHEN, JL KEY 1975 Isolation of nucleoli andlocalization of ribonucleic acid polymerase I from soybean hypocotyl. PlantPhysiol 56: 850-852

20. LIN CY, YM CHEN, TJ GUILFOYLE, JL KEY 1976 Selective modulation ofRNA polymerase I activity during growth transitions in the soybean seedling.Plant Physiol 58: 614-617

21. LOWRY, OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1961 Protein meas-urement with the Folin phenol reagent. J Biol Chem 192: 265-275

22. MCCONKEY EH, JW HOPKINS 1964 The relationship of the nucleolus to thesynthesis of rRNA in HeLa cells. Proc Natl Acad Sci USA 51: 1197-1204

23. MELANSON DL, J INGLE 1978 Regulation of ribosomal RNA accumulation byauxin in the artichoke tissue. Plant Physiol 62: 761-765

24. MErS L, L BOGORAD 1974 Two-dimensional polyacrylamide gel electropho-resis: an improved method for ribosomal proteins. Anal Biochem 57: 200-210

25. ROEGER ME, UE LOENING, RSS FRASER 1970 Ribosomal precursor in plants.J Mol Biol 49: 681-692

26. SCHNEIDER WC 1957 Determination of nucleic acid by pentose analysis.Methods Enzymol 3: 680-684

27. SPIKER S, JL KEY, B WAKIN 1976 Identification and fractionation of planthistones. Arch Biochem Biophy 176: 510-518

28. TAKAHASHI N, J SHIMADA, J HIGO, S SIGo, S TANIFUGI 1972 Ribosomalprecursor particles from plant nucleoli. Biochim Biophys Acta 262: 502-513

29. WARNER JR, R SOEIRO 1967 Nascent ribosomes from HeLa cells. Proc NatlAcad Sci USA 58: 1984-1990

30. WARNER JR 1974 The assembly of ribosomes in eukaryotes. In M Nomura, ATissieres, P Lengyel, eds, The Ribosomes. Spring Harbor Laboratory, ColdSpring Harbor, New York, pp 461-488

31. WEINBERG RA, S PENMAN 1970 Processing of 45S nucleolar RNA. J Mol Biol47: 169-178

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