Plant Physiol. (1985) 77, 99-1030032-0889/85/77/0099/05/$0 1.00/0

Cytokinin-Modulated Gene Expression in Excised PumpkinCotyledons'

Received for publication July 24, 1984

CHONG-MAW CHEN* AND Scorr M. LEISNERDepartment ofLife Science and Biomedical Research Institute, University of Wisconsin-Parkside,Kenosha, Wisconsin 53141

ABSTRACT

Comparison of two-dimensional polyacrylamide gel electrophoreticmaps of proteins isolated from benzyladenine-treated and untreatedpumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that theexpression of certain proteins is enhanced, induced, or suppressed by thecytokinin treatment. The amount of poly(A)' mRNA isolated from coty-ledons incubated with 10' molar benzyladenine for five days was aboutfour-fold over the water-incubated control. The activity of hydroxypyru-vate reductase prepared from purified cotyledonous microbodies andanalyzed by native gel electrophoresis is proportionally enhanced bysequentially higher concentrations (10' to 101 molar) of benzyladenine.Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxy-pyruvate reductase activity, thus, the enzyme synthesis does not appearto be controlled by organelie genes. Hydroxypyruvate reductase synthesisis inhibited by cycloheximide, cordycepin, and to a certain degree byactinomycin D. These data support the view ofa close association betweencytokinin action and gene expression.

Cytokinins promote the development of excised cotyledons ofvarious plants, particularly in darkness. The most importantresponses of the cotyledons to the hormonal stimulation are cellexpansion, breakdown of storage material (9, 11, 12), wateruptake (14), increase in fresh weight, and the development ofcell organelles (7, 12). This wide range of responses is accompa-nied by an increment ofDNA synthesis (8), induction ofproteins,or enzymes synthesis (9, 12). One of the enzyme activitiessignificantly stimulated by the hormone is HPR2 (12). Thisenzyme catalyzes the conversion ofhydroxypyruvate to glyceratein microbodies, and is involved in the glycolate pathway (16).

Despite an abundance of knowledge about the physiologicalresponses of cotyledons to cytokinin treatment, there is noconcrete information on the questions of (a) whether the effectsof cytokinin on gene expression are exerted by induction, stim-ulation, and/or suppression of specific protein syntheses, and (b)if the influence of cytokinin on protein or enzyme synthesis isprimarily at the pretranslational or posttranslational level.

In the present study, we have utilized two-dimensional PAGEto determine if cytokinin modifies the spectrum of polypeptidessynthesized during cytokinin-induced cell expansion ofpumpkincotyledons. We have also employed inhibitors to analyze whetherthe enhancement ofHPR production modulated by a cytokininis at the transcriptional or translational level.

I Supported by the National Science Foundation PCM-8204717 toC-M. C.

2Abbreviation: HPR, hydroxypyruvate reductase.

MATERIAIS AND METHODS

Chemicals and Plant Materials. Antibiotics, N6-benzylade-nine, Carbowax, hydroxypyruvic acid, DL-glycerate, NAD, andNADH were obtained from Sigma; Acrylamide and bisacryl-amide were purchased from Bio-Rad; ammonium persulfate wasa product of Fisher. Pumpkin (Cucurbita pepo L. cv Halloween)seeds were obtained from Olds Seed Co. (Madison, WI).

Cotyledon Preparation. Pumpkin seeds of approximately thesame size were imbibed for 16 h with running tap water, andsurface sterilized for 10 min with 0.5% NaOCl. The seeds werethoroughly rinsed with sterile distilled H20 and were sown ondistilled H20-moistened Whatman No. 1 filter papers containedin Petri dishes under aseptic conditions. These Petri dishes wereplaced in darkness at room temperature (25°C) for 7 d. Cotyle-dons were then excised and cultured under aseptic conditionsfor various times in the dark on filter papers in Petri dishes. Thedishes contained 10 ml distilled H20, 0.2 mg/ml penicillin G,with or without BA.

Preparation of Microbody HPR. The preparation of pumpkincotyledon microbodies was adapted from a method for spinachmicrobody preparation described by Huang et al. (10) and mod-ified by Chang and Huang (3) with the exception that thehomogenate was filtered through three layers of cheeseclothinstead of Nitex cloth. The microbodies were purified by a 30 to60% (w/v) linear sucrose gradient. Sucrose gradient-purifiedmicrobodies were dialyzed against 10 mm Tris-HCI (pH 7.5)overnight to release proteins from microbodies. The dialysatewas centrifuged at 100,000g for 1 h, and the supernatant proteinsolution containing HPR was reduced to about 1 ml by Carbo-wax. All procedures were conducted at 0 to 4°C. This crudeenzyme preparation was analyzed for HPR activity by nonde-naturing gel electrophoresis (4) or was purified further by DEAE-52 cellulose chromatography (1.75 x 109.5 cm).

Protein concentration was determined by the assay ofBradford(2). Column effluents were monitored photometrically (at 280nm) for protein. The density of sucrose gradient fractions wasdetermined from measurements of their refractive indices.Enzyme Assays. HPR activity was assayed by spectrophoto-

metric method based on the decrease in absorption at 340 nm(16). The assay mixture contained 20 mm Na-phosphate (pH6.2), 1.5 mM hydroxypyruvic acid, 0.2 mm NADH, and 10 to 50,ul of enzyme preparation in a total volume of 0.8 ml. A Carymodel 14 was used to quantify optical density. One unit ofHPRactivity is defined as the quantity of enzyme which will catalyzethe oxidation of 1.0 Mmol ofNADH per min in a standard assay.

Lactic dehydrogenase activity was assayed by the same reactionmixture for HPR except that lactate and NAD were used toreplace hydroxypyruvate and NADH.In Situ HPR Assay. Native polyacrylamide gels for the in situ

assay of HPR were prepared (4) and the enzyme activity wasdetected by a staining method adapted from the procedure of

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Plant Physiol. Vol. 77, 1985

Dewey and Conklin (5). The reaction is performed in the direc-tion of glycerate oxidation and results in the appearance ofreduced nitro blue tetrazolium in the gel as a pinkish-purpleprecipitate. Immediately after electrophoresis, the native gel wasplaced in the in situ assay solution and stained in the dark for60 min at 37°C. The in situ assay solution contained 250 mMTris-HCI (pH 8.5), 50 mM DL-glycerate, 0.6 mm nitro bluetetrazolium, 2 mm NAD, 0.4 mm phenazine methosulfate. Thereaction was stopped by addition of 7% acetic acid. Controlswere performed in the absence of glycerate or NAD duringstaining.

Electrophoretic Procedures. Native PAGE was performed us-ing 1.5-mm thick slab gels. The gels were casted with 7% acryl-amide and the acrylamide:bisacrylamide ratio was 38:1. Toprevent shrinkage during electrophoresis, the gels were preparedin a cold room (about 4C) and electrophoresed at low temper-ature (10°C) using LKB 2209 multitemp cooling system. Two-dimensional PAGE with isoelectric focusing in the first dimen-sion and SDS in the second dimension was performed as de-scribed by O'Farrell (13).

RESULTS

Effect of BA on Fresh Weight Yield, mRNA, and ProteinConcentrations. In the absence of light, the growth of excisedpumpkin cotyledons is markedly enhanced by a cytokinin treat-ment. Time course studies of fresh weight yield and proteinconcentration in the excised cotyledons treated with 101 M BAare shown in Figure 1A. The fresh weight increment of BA-treated cotyledons on day 5 is about 2-fold over the water-treatedones. However, there was no significant increase in cotyledonsdry weight for the BA-treated (5.3 g/100 cotyledons) as comparedto the untreated controls (5.2 g/100 cotyledons).The protein concentration of cotyledons treated with 10-4 M

BA for 5 d is about 45% greater than that of water-treatedcontrols (Fig. IA). The increment in protein concentration maybe due to an enhanced rate of protein synthesis and/or increasedconversion of other macromolecules (such as lipid and carbo-hydrate) to protein. The protein concentration (,ug/cotyledon)

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FIG. 1. Time course of increase in fresh weight and protein concen-tration (A) and hydroxypyruvate reductase (HPR) activity (B) of excisedpumpkin cotyledons. The cotyledons were incubated with 10' M BA orwater. Each treatment represents 200 cotyledons grown in the dark atroom temperature.

decreases significantly in BA-treated cotyledons after 5 d ofincubation. The reasons for the decrease are unknown. It maybe caused by the depletion ofprotein synthetic precursors during5 d of BA treatment. Comparison of poly(A)+mRNA isolatedfrom cotyledons incubated with 10' M BA or water alone for 5d, and purified on oligo (dT)-cellulose column (1), showed thatpoly(A)+mRNA content in Ba-treated cotyledons was 4-fold overthe water-treated ones (BA-treated: 10 ,ug poly(A)+mRNA/gcotyledons; water-treated: 2.5 ,ug poly(A)+mRNA/g cotyledons).According to the time course studies of protein content and

HPR activity (Fig. 1), the duration of the hormonal or watertreatment which produced maximal differences in protein con-centrations and HPR activity is 3 to 5 d. Therefore, this periodof incubation was chosen to study the two-dimensional poly-acrylamide gel electrophoretic maps ofproteins and the enhance-ment of HPR activity by treatment with various concentrationsof BA. Our primary goal of this study is to demonstrate a varietyof macromolecular events which occur after treatment of thecotyledons with cytokinin. Thus, the syntheses of short-livedmRNAs and proteins which respond to cytokinin treatmentsneed a separate investigation.

Cytokinin-Induced Changes in the Population of Proteins. Ifthe synthesis of specific polypeptides required for cell expansionis enhanced or induced by cytokinin, we might expect a differ-ence in the polypeptide population between untreated and cy-tokinin-treated cotyledons. Thus, the spectrum of total solublecell proteins accumulated in excised pumpkin cotyledons incu-bated with or without 10-4 M BA was analyzed by two-dimen-sional PAGE (Fig. 2). The periods ofBA incubation ranged from1 to 6 d in the dark. BA appears to induce, enhance, reduce, andsuppress the expression of a number of specific polypeptides inthe expanding cotyledons. After 4-d incubation among the poly-peptide spots modulated by cytokinins, about 14 are induced, 85are enhanced, 5 are reduced, and 5 are suppressed. The differ-ences in polypeptide populations are probably not due to proteasedegradation of protein because protease inhibitors (1 mm phen-ylmethyl-sufonylfluoride and 2.5 mM N-methylmaleimide) werepresent during the process of protein preparation.Enhancement of Microbody Hydroxypyruvate Reductase by

Benzyladenine. One of the proteins enhanced by BA is HPR.The enzyme was isolated from sucrose gradient-purified micro-bodies. The activity of microbody HPR prepared from cotyle-dons treated with 10-4 M BA for 5 d was enhanced about 4-foldover the water-treated control (Fig. 1 B). Enhanced HPR activityis likely due to more HPR in BA-treated microbodies, becausethe HPR activity was measured on the basis of the amount ofNADH oxidized/mg microbody protein.The microbody HPR prepared from cotyledons treated with

10-4 M BA for 4 d was further purified by a DEAE-52 cellulosecolumn (1.75 x 109.5 cm) equilibrated with 10 mm Tris.HClbuffer (pH 7.5). The HPR was eluted with a continuous gradientof NaCl (0-0.2 M) in the same buffer, and the enzyme wasseparated by the column into two major activity fractions (bedvolume: F-1, 1.75) (0.12 mM CIXO.14 M NaCl); F-2, 2.18). HPRobtained from water-treated control had a similar elution profileto that of the BA-treated one, but the HPR activity/mg ofmicrobody protein was about 25% that of the enzyme preparedfrom BA-treated cotyledons.Optimal pH values for enzyme activity ofeach enzyme fraction

were determined by using either hydroxypyruvate or glycericacid (for reverse reaction) as a substrate. The pH optimumdepends upon the enzyme fraction and substrate used; pH op-tima for the F-l fraction are: 6.3 to 6.5 (hydroxypyruvate as asubstrate) and 8.1 to 8.3 (glyceric acid as a substrate for reversereaction); for the F-2 fraction are: 7.2 to 7.5 (hydroxypyruvate)and 8.4 to 8.6 (glyceric acid). These results also indicate thatthese two enzyme fractions are isozymes.

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100 CHEN AND LEISNER

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CYTOKININ-MODULATED GENE EXPRESSION

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FIG. 2. Two-dimensional polyacrylamide gel electrophoretic maps of proteins extracted from excised pumpkin cotyledons that were incubatedfor 4 d in the dark in the presence (A) or absence (B) of 101 M BA. The first dimension was loaded with 80 ,g of protein and the proteins separatedby SDS gel in the second dimensions were visualized by Bio-Rad (Richmond, CA) silver stain. Enhanced (0); induced ( ); reduced (A); supressed(-).

To examine whether certain HPR isozymes are differentiallyenhanced by BA treatment of cotyledons, total microbody HPRisozymes were analyzed by nondenaturing gel electrophoresis.Figure 3 shows the increase ofHPR isozyme activity by treatmentof pumpkin cotyledons with various BA concentrations (10- to10' M) for 3 d. The intensity of the isozyme activity, measuredby densitometry, is proportional to the concentration of BAapplied to the cotyledons. The increase of the isozyme activityby 10-' M BA is about 4-fold over the 10-' M BA treated ones,and about 5-fold over the water-treated control. Furthermore, allof the isozymes are uniformly enhanced by the hormone. Thehighest concentration of BA applied to the cotyledons are 10-4M which is the upper limit of BA solubility in water. Therefore,it is unknown whether higher BA concentrations (10-3 M orgreater) would result in even more pronounced responses. Wealso do not know the extent of BA uptake by the cells ofcotyledons.

Addition of BA (10-8_10-4 M) to a cell-free enzyme reactionmixture containing HPR did not affect the enzyme activity; thus,HPR activity is not directly influenced by BA in a cell-freesystem.Examination of HPR activity in the cytosol indicated that

about equal amounts of HPR were present in cytosol as inmicrobodies. However, it is not clear whether the cytosolic HPRwas released from microbodies during enzyme preparation.

Effect of Inhibitors on Cytokinin-Enhanced HydroxypyruvateActivity. Experiments were designed to investigate whether cy-tokinin-enhanced HPR activity is regulated by the hormone atthe transcriptional or translational level. Results in Table I andII show that cotyledon expansion and HPR activity are bothinhibited, to different degrees, by transcriptional and transla-tional inhibitors. Cytokinin-enhanced HPR activity is inhibited82% by cordycepin (10' M) and 60% by antinomycin D (20 ,g/ml) after 4 d of treatment, thus implying that the inhibition ofBA-enhanced HPR activity may be at the transcriptional level.Reasons for the differences in HPR inhibitory activity by cor-

dycepin and actinomycin D are unknown; it may be due tovariation in the permeability of cotyledons to these inhibitors.The data from cycloheximide experiments show that this anti-biotic (10 ug/ml) inhibited 59% of BA-enhanced expansion and95% of BA-enhanced HPR activity. However, it is not clearwhether cycloheximide blocks the synthesis ofparticular proteinswhich are required for the induction of specific mRNAs relatedto cell expansion and HPR synthesis, or inhibits the posttran-scriptional processes regulated by the cytokinin. Incubation ofthe cotyledons for 4 d in the presence of20 ,g/ml ofactinomycinD reduced significantly the induced and enhanced polypeptidespots in 2-D gel analyses, while 10-' M cordycepin reduced almostevery polypeptide spot which appeared on the 2-D gels (resultsnot shown).Ethidium bromide at the concentration of 1 gg/ml has been

shown to inhibit mitochondrial RNA synthesis but not the RNAsynthesis associated with the nucleus (6). When cotyledons wereincubated with ethidium bromide (1 ;g/ml) or H20 (control) for3 d, ethidium bromide did not affect HPR activity, which impliesthat the HPR gene is not located in the mitochondria.

DISCUSSION

When we initiated the research described in this report, weposed two questions. First of all, does cytokinin simply maintainor enhance general protein synthesis in excised organs, or doescytokinin alter a limited number of specific polypeptides? Sec-ond, is the influence of cytokinin on the synthesis of a particularenzyme, such as HPR, at the pretranslational and/or posttrans-lational level?The first question is partly answered by 2-D gel analyses ofthe

spectrum ofpolypeptides which accumulated in excised pumpkincotyledons incubated in the presence or absence of a cytokinin,BA (Fig. 2). The 2-D gel data show both quantitative andqualitative changes in cotyledon proteins during BA-mediatedchanges in cotyledon growth. The synthesis of certain proteinsappear to be induced or suppressed, while others are enhanced

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Plant Physiol. Vol. 77, 1985

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Table I. Effect ofInhibitors on Benzyladenine-Enhanced Expansion ofPumpkin Cotyledons

Pumpkin seeds of approximately the same size (200 seeds for eachtreatment) were imbibed for 16 h with water, and surface-sterilized for10 min with 0.5% NaOCI. The seeds were thoroughly rinsed with steriledistilled H20, and grown on sterile filter paper in Petri dishes in the darkfor 7 d. Cotyledons were excised and incubated in the solution contained10' M BA with or without inhibitor under aseptic conditions for 4 d inthe dark.

Fresh Wt. YieldInhibitor Concn. With No Inhibition

inhibitor inhibitorg/100 cotyledons %

Cordycepin I mM 18.4 27.1 32Cycloheximide 10 ,g/ml 12.8 31.3 59Actinomycin D 20 1g/ml 24.7 28.9 15Ethidium bromide I 'Ug/ml 29.3 29.5 0

by the presence of BA. However, the functional identities of thecytokinin-regulated proteins, with the exception ofHPR, are notknown. We also do not know what factors are involved ininduction, suppression, and enhancement of gene expression

FIG. 3. Enhancement of hy-droxypyruvate reductase activityby various concentrations ofbenzyladenine. Excised pump-kin cotyledons were incubatedwith indicated concentrations ofBA or water for 3 d, and HPRprepared from sucrose gradient-purified microbodies was electro-phoresed on a nondenaturinggel. The activity ofHPR was de-tected by in situ assay as de-scribed in the text. Top panel,densitometric scanning of thedensities of BA-enhanced HPRisozymes. The numbers I to IVcorrespond to the isozyme bandsI to IV shown in the bottompanel. Bottom panel, lane a, lac-tic acid was used to replace gly-ceric acid as a substrate for theenzymic reaction; lane b, glycericacid, a substrate for the enzyme,was not included. The concen-trations (104-104 M) ofBA rep-resent cotyledons preincubatedwith indicated concentrations ofBA for 3 d.

Table II. Effect ofInhibitors on Benzyladenine-EnhancedHydroxypyruvate Reductase Activity

Preparation of pumpkin cotyledons (200 per treatment) were as de-scribed in Table I. HPR was isolated from sucrose gradient-purifiedmicrobodies and was not purified further. The enzyme activity fromcotyledon incubated with water alone was 0.75 umol NADH oxidized/mg microbody protein. min.

Inhibitor Concn. HPR Activity Inhibitionjimol NADH oxidized/mg %microbody protein- min

None (control) 3.75 0Cordycepin 1 mm 0.68 81.9Cycloheximide 10 ,g/ml 0.16 95.6Actinomycin D 208/ml 1.49 60.0Ethidium bromide I gsg/ml 3.81 0

modulated by the hormone. Understanding of these factors isimportant in elucidating cytokinin-regulated plant growth. Oneofthe enzymes markedly enhanced by cytokinin is HPR. Longoet al. (12) have made similar observations using excised water-melon cotyledons. The expression ofthis HPR gene is modulatedby different concentrations of BA (Fig. 3). Thus, HPR shouldserve as an excellent model system in characterizing the pre-

102 CHEN AND LEISNER

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CYTOKININ-MODULATED GENE EXPRESSION

translational regulation of an enzyme by a hormone.Auxins, another group of plant hormones, have been shown

to induce changes in the level of translatable mRNAs (for ex-amples 15, 17, 18). Our preliminary results (not shown) alsoindicate that BA is capable of inducing changes in the level oftranslatable poly(A)+ mRNA isolated from excised pumpkincotyledons.The question of whether cytokinin affects gene expression at

the transcriptional or translational level cannot be clearly an-swered by the inhibitor studies. Nevertheless, BA-enhanced ex-pansion ofpumpkin cotyledons, as measured by increase in freshweight, was inhibited 32% by 10-3 M cordycepin and 15% by 20g/ml actinomycin D (Table I). Furthermore, BA (10-4 M) in-creased the amounts of poly(A)+mRNA about 4-fold in thecotyledons, and BA-enhanced HPR activity was markedly in-hibited by cordycepin as well as actinomycin D (Table II). Thesedata taken together are suggestive of a requirement for at leastsome RNA syntheses in the cytokinin-induced growth. It is notclear if cytokinin mobilizes stored mRNAs. At present, we alsocannot offer a satisfactory explanation for the differences betweencotyledon expansion and HPR activity exerted by the inhibitors.From the experimental data of Table II, it is likely that theenhancement of HPR synthesis by BA is regulated at the tran-scriptional level. To unequivocally determine whether cytokininregulates the synthesis of HPR and other proteins at the tran-scriptional level, in vitro synthesis of proteins and two-dimen-sional gel analyses of these proteins are needed. Alternatively,DNA clones corresponding to HPR or other proteins can be usedas probes to investigate cytokinin and gene expression.

LITERATURE CITED

1. Aviv H, P LEDER 1972 Purification of biologically active globin messengerRNA by chromatography on oligothymidylic acid-cellulose. Proc Natl AcadSci USA 69: 1408-1412

2. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye bind-ing. Anal Biochem 72: 284-264

3. CHANG CC, AHC HUANG 1981 Metabolism of glycolate in isolated spinachleaf peroxisomes. Plant Physiol 67: 1003-1006

4. DAVIS BJ 1964 Gel for nondenature gel electrophoresis. Part II. Clinicalapplications. Method and application to human serum proteins. Ann NYAcad Sci 121: 405-427

5. DEwEy MM, JL CONKLIN 1961 Starch gel electrophoresis of lactic dehydrog-enase from rat kidney. Proc Soc Exp Biol Med 105: 492-494

6. FAN H, S PENMAN 1970 Mitochondrial RNA synthesis during mitosis. Science168: 135-138

7. FANTELLI R, GP LONGO, G Rossi, CL LONGO, B VITELLI 1984 Interactionbetween benzyladenine and fusicoccin on the development ofexcised water-melon cotyledons. I. Growth and organelle development. Plant Science Lett33: 269-275

8. GALLI MG 1984 Synthesis of DNA in excised watermelon cotyledons grownin water and benzyladenine. Planta 160: 193-199

9. HOWARD HF, FH WITHAM 1983 Invertase activity and the kinetin-stimulatedenlargement of detached radish cotyledons. Plant Physiol 73: 304-308

10. HUANG AHC, KDF Liu, RJ YOULE 1976 Organelle specific isozymes ofaspartate-alpha-ketoglutarate transaminase in spinach leaves. Plant Physiol58: 110-113

1 1. HUFF A, CW Ross 1975 Promotion of radish cotyledon enlrgement andreducing sugar content by zeatin and red light. Plant Physiol 56: 429-432

12. LONGO CP, GP LONGO, MG LAMPUGNANI, G Rossi, 0 SERvErrAz 1981 Lightand fusicoccin as tools for discriminating among responses of cotyledons tocytokinins. In J Guern, C Peaud-Lenoel, eds, Metabolism and MolecularActivities of Cytokinins. Springer, New York, pp 261-266

13. O'FARRELL PH 1975 High resolution two-dimensional electrophoresis of pro-teins. J Biol Chem 250: 4007-4021

14. RAYLE DL, CW Ross, N RoBINsoN 1982 Estimation of osmotic parametersaccompanying zeatin-induced growth of detached cucumber cotyledons.Plant Physiol 70: 1634-1636

15. THEOLOaGS A, PM RAY 1982 Early auxin-regulated polyadenylated mRNAsequences in pea stem tissue. Proc Natl Acad Sci USA 79: 418421

16. TOLBERT NE, RK YAMAZAKI, A OESER 1970 Localiztion and properties ofhydroxypyruvate and glyoxylate reductase in spinach leaf particles. J BiolChem 245: 5129-5136

17. WALKER JC, JL KEY 1982 Isolation of cloned cDNAs to auxin-responsivepoly(A)'RNAs of elongating soybean hypocotyl. Proc Natl Acad Sci USA79: 7185-7189

18. ZURFLUH LL, TJ GUILFOYLE 1982 Auxin-induced changes in the populationof translatable messenger RNA in elongating sections of soybean hypocotyl.Plant Physiol 69: 332-337

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