Journal of Experimental Botany, Vol. 35, No. 155, pp. 882-889, June 1984

Differential Effect of Ribosome-Inactivating Proteins on

Plant Ribosome Activity and Plant Cells Growth

MARIA GIULIA BATTELLI, ENZO LORENZONI ANDFIORENZO STIRPE

Istituto di Patologia Generate dell'Universita di Bologna, 1-40126 Bologna, Italia

RINO CELLA AND BRUNO PARISI1

Dipartimento di Genetica e Microbiologia, Sezione di Microbiologia e Fisiologia Vegetaledell'Universita di Pavia, 1-27100 Pavia, Italia

Received 3 1 October 1983

ABSTRACTThe effect of ribosome-inactivating proteins (RIPs), either single-chain or toxins, was studied on plantribosomes. RIPs did not affect ribosomes from their own plants, while inhibiting to a variable extentprotein synthesis by heterologous plant ribosomes. Ricin stimulated and PAP-S inhibited the growth ofcarrot cells in culture.

Key words: Plant ribosomes; Ribosome-inactivating proteins; Protein synthesis; Ribosome specificity:Plant cell cultures.

I N T R O D U C T I O NRibosome-inactivating proteins are present in many and possibly all plants, although in highlyvariable concentrations (Barbieri and Stirpe, 1981). They exist either as single-chain proteins,or as A (active) chains linked to a B (binding) chain in toxins (ricin, abrin, modeccin,viscumin) (Olsnes and Pihl, 1982). Common properties of RIPs are (i) the capacity ofinactivating the 60 S subunit of eukaryotic ribosomes, and (ii) the inhibition of replication ofplant viruses. The latter property was attributed to easier penetration of RIPs intovirus-infected cells, with consequent inactivation of ribosomes, arrest of protein synthesis, andinhibition of virus replication (Owens, Bruening and Shepherd, 1973). This antiviral activityis exerted only on plants different from those from which RIPs derive. Further, one of theRIPs, the pokeweed antiviral protein (PAP), inactivates wheat germ and cowpea ribosomesbut not pokeweed ribosomes (Owens el ai, 1973), and tritin, an RIP from wheat germ, doesnot affect wheat-germ ribosomes (Coleman and Roberts, 1981). These observations led to thehypothesis that RIPs should act only on heterologous ribosomes (Stirpe, 1982).

1 Correspondence to Professor Bruno Parisi, Dipartimento di Genetica e Microbiologia. Via S. Epifanio. 14.1-27100 Pavia, Italia.

2 Abbreviations: MCI, Momordica charantia inhibitor; PAP, pokeweed antiviral protein; PAP-S. pokeweedantiviral protein from seeds; RIP(s), ribosome-inactivating protein(s).

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Baltelli et al.—Ribosome-Inactivating Proteins and Plant Ribosomes 883

Present experiments are in support of this notion. RIPs that could be tested on homologousribosomes did not affect them significantly, while inactivating to a variable extent those fromheterologous species. It was observed also that PAP inhibited, and ricin stimulated, thegrowth of a carrot cell line in liquid culture.

M A T E R I A L S AND M E T H O D SR ibosome-inactivating proteinsSingle-chain RIPs. prepared as described in the respective references, were: PAP-S from Phytolaccaamericana (pokeweed) (Barbieri. Aron. Irvin and Stirpe. 1982). gelonin from Gelonium multijlorum(Stirpe. Olsnes and Pihl. 1980). the inhibitor from Momordica charantia (MCI) (Barbieri. Zamboni.Lorenzoni. Montanaro. Sperti and Stirpe. 1980). and dianthin 32 from Dianthus caryophyllus(carnation) (Stirpe. Williams. Onyon. Legg and Stevens. 1981). Toxins were: ricin from Ricinuscommunis (castor bean) (Nicolson. Blaustein and Etzler. 1974). abrin C from Abrus precatorius(Jequirity bean) (Wei. Hertman. Pfuderer and Wang. 1974). modeccin 4B from Adenia digitata(Gasperi-Campani. Barbieri. Lorenzoni. Montanaro. Sperti. Bonetti and Stirpe. 1978; Barbieri.Zamboni. Montanaro. Sperti and Stirpe. 1980) and viscumin. the toxin from Viscum album (mistletoe)(Ziska. Franz and Kindt. 1978).

For the experiments with ribosomes. toxins were reduced by incubation at 37 °C for 2 h with 1%2-mercaptoethanol. a treatment that enhances the inhibitory effect on cell-free systems, whilstdecreasing toxicity to intact cells (Olsnes and Pihl. 1972).

R ibosomesR. communis leaves (20 g) were ground in a pre-chilled mortar with 10 g of quartz sand and 6 vols of

300molm ' Tris/HCI buffer. pH 8-5. containing 250molm •' sucrose. 50 mol m~3 MgCU, lOmolm"'dithiothreitol and 0-1 mol m~3 EDTA. All other leaves and wheat germ were ground with 3 vols of100 mol m"1 potassium Bicine buffer. pH 8-0. containing the components listed above except EDTA.Rat livers were homogenized in a Potter homogenizer with 3 vols of the latter buffer. Carrot and ricecells (2 0 g. wet wt.) were homogenized as livers, but with 2 vols of Tris/HCI buffer. pH 7-4. containing2-0 mol m ; magnesium acetate. 10 mol m~' 2-mercaptoethanol. and 0-1 mol m~' EDTA.Ribosomes were isolated by centrifugations and were resuspended as described (Owens et al., 1973).After the first centrifugation. carrot cell homogenates were adjusted to 250 mol m~' sucrose.Poly(U)-directed polyphenylalanine synthesis was determined as described (Montanaro, Sperti.Zamboni. Denaro. Testoni. Gasperi-Campani and Stirpe. 1978). In all cases S-100 rat-liversupernatant (Staehelin and Falvey. 1971) was used as a source of soluble enzymes.

Cell culturesDaucus carota (carrot) and Oryza saliva (rice) liquid cell cultures were grown in MS medium

(Murashige and Skoog. 1962) containing 0-5 mg dm"3 of 2.4-dichlorophenoxyacetic acid and 0-25 mgcm"' of 6-benzylaminopurine. or in R2 medium (Ohira. Ojima and Fujiwara, 1973), respectively. Twocarrot cell lines were tested: Cl and the embryogenetic line E2 (Nielsen. Rollo. Parisi. Cella and Sala.1979). In the experiments without ammonium ion. ammonium salts were omitted from the growthmedium (carrot cultures), or were substituted by an equimolar amount of potassium nitrate andpotassium sulphate (rice cultures). The effect of RIPs on the growth of cell cultures was determinedessentially as described (Nielsen el al.. 1979). except that cells were inoculated at 0-5 mg cm"3 (drywt.).

RESULTSThe effect of RIPs on polyphenylalanine synthesis by ribosomes from various plant specieswas examined preliminarily at the concentration of 100 fig cm"3 (0-033 mol m~3 and1-7 mmol m"3, respectively for single-chain proteins and for toxins) (Table 1). When aninhibition greater than 30% was observed, dose-response curves were obtained, from whichthe ID50's (concentrations causing 50% inhibition) were calculated by the linear regressionmethod. These are reported in Table 2, together with the ID50's upon rat-liver ribosomes. Thedose-response curves usually were of the type shown in Fig. 1; however, in the case ofPAP-S on rice cell ribosomes a maximum inhibition of 50% was obtained, which was notenhanced by further increasing the concentration of PAP—S (Fig. 2).

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884 Battelli et al.—R ibosome-Inactivating Proteins and Plant R ibosomes

T A B L E 1. Effect of ribosome-inactivating proteins on polyphenylalanine synthesis by

ribosomes from various plant species

Ribosomes from

Dianthus caryophyllus(carnation) leaves

Phytolacca americana(pokeweed) leaves

Nicotiana tabacum(tobacco) leaves

Ricinus communis(castor bean) leaves

Triticum aeslivum"(wheat germ)

Daucus carota(carrot) leavescells E2 line

C 1 line"Oryza sativa

(rice) cells

Ribosome-inactivating protein added

MCI

17

68

62

96

15

89

211835

Dianthin32

86

84

2

27

15

26

333233

Gelonin PAP-S

Polyphenylalanine

96

89

85

94

69

105

807266

86

110

0

37

26

42

202842

1 (100//gem"3)

Ricin Abrin Modeccin

synthesis (% of control)

97

117

96

92

102

103

1168999

117

123

17

67

69

50

424691

117

117

100

100

106

92

11611697

Viscumalbum toxin

99

119

80

105

98

98

122112100

Reaction mixtures contained, in a final volume of 0-1 cm1: 80 mol m~} Tris/HCl buffer, pH 7-4,120 mol m~3 KC1, 7-0 mol m~-' magnesium acetate, 2-0 mol rrr3 dithiothreitol, 2-0 mol m~} GTP,80//g of poly(U), 10 mm3 of S-100 rat-liver supernatant, 14C phenylalanyl-tRNA (5000 d.p.m., exceptwhen otherwise stated) and ribosomes as indicated in Table 2. After incubation at 24 °C for 30 min thereaction was arrested and the hot-trichloroacetic-acid insoluble radioactivity was measured asdescribed by Montanaro el al. (1978); values of zero-time blanks were subtracted. Control values weresimilar to those reported in Table 2.

" 9900 d.p.m. of phenylalanyl-tRN A added.'' 13 200 d.p.m. of phenylalanyl-tRNA added.

T A B L E 2. Inhibitory activity of ribosome-inactivating proteins on polyphenylalaninesynthesis

Ribosomes from

Rat liverCarnation leavesPokeweed leavesTobacco leavesCastor bean leavesWheat germ"Carrot leaves

E2 line cellsC1 line cells*

Rice cells

Ribosomes added(mg/crrr3)

0-40-73-71-63-00-153-30-10-20-2

Control values(d.p.m.)

18051236444

1573718

6465531

365578804767

MCI

0-341

230140

38

23-52860

Dianthin 32(ID50 fig cm3)

0-7

4 07-810

13-50-30-44.2

PAP-S

0-3

2-5370-55

641-52-34-5

Abrin

0-4

271601271007593

Experimental conditions were as in Table 1." 9900 d.p.m. of phenylalanine-tRNA added.* 13 200 d.p.m. of phenylalanyl-tRNA added.

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Battelli et al.—R ibosome-Inactivating Proteins and Plant R ibosomes

1001

885

Abrin concentration (/ug cm 3)

FIG. 1. Effect of abrin on polyphenylalanine synthesis by tobacco leaf ribosomes. Experimentalconditions were as described in Table 1. The amount of ribosomes used and control values were as in

Table 1.

-+ •0 50 100

PAP-S concentration (fig cm"3)

FIG. 2. Effect of increasing concentrations of PAP-S on polyphenylalanine synthesis by rice cellribosomes.

Those RIPs that could be tested on homologous ribosomes (dianthin 32, PAP-S and ricin,respectively on ribosomes from carnation, pokeweed and castor bean) did not significantlyaffect their capacity to synthesize polyphenylalanine. The effect of RIPs on heterologousribosomes varied considerably both from one RIP to another, and from species to species of

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886 Battelli et al.—R ibosome-Inactivating Proteins and Plant R ibosomes

100-

3 50 ••

cj

10 100 300 10RIPs concentration (fig cm"3 cell culture)

100 300

FIG. 3. Effect of RIPs on the growth of carrot and rice liquid cell cultures. Cells are grown as 10 cm3

aliquots in 50 cm3 flasks in the presence of RIPs at the indicated concentrations. Cell growth wasdetermined as increase of dry weight and was expressed as % of the growth of controls, after

substracting the value of the inoculum. • — • Ricin; © — © PAP-S; O—Ogelonin.

ribosomes. Thus carnation and pokeweed ribosomes were sensitive only to high con-centrations of MCI, whereas other ribosomes were inhibited to different extents by diathin 32,MCI, PAP-S and abrin. Gelonin had a moderate effect on ribosomes from wheat-germ andfrom a carrot cell line, whereas ricin, modeccin and viscumin at the concentrations used didnot affect significantly any of the plant ribosomes examined. In most cases the effect of RIPswas much more marked on rat liver than on plant ribosomes.

The effect of some RIPs on the growth of rice and carrot E2 cell lines in liquid cultures wastested. The growth of carrot cells was markedly reduced by PAP-S (ID50 50 n% cm~\approximately), whereas gelonin had no effect, and ricin actually stimulated growth (Fig. 3).The same RIPs did not affect, and possibly stimulated, the growth of rice cells. The figureshows the results obtained in the absence of ammonium ions, which reduce the toxicity ofmodeccin to HeLa cells (Sandvig, Olsnes, and Pihl, 1979). Similar results were obtained bothfor carrot and rice cells in the presence of ammonium ions, and for carrot Cl line (results notshown).

D I S C U S S I O NOur results demonstrate that RIPs have a more marked effect on mammalian than on plantribosomes, as observed already with ricin (Cawley, Hedblom, Hoffman and Houston, 1977;Cawley, Hedblom and Houston, 1979; Lugnier and Rether, 1981; Harley and Beevers,1982). The lack of effect of RIPs on homologous ribosomes is consistent with the lack ofinhibition of pokeweed (Owens et al., 1973) and wheat-germ ribosomes (Coleman, andRoberts, 1981) by PAP and tritin, respectively. Our results are consistent with those ofReisbig and Bruland (1983), who observed that dianthins inactivate purified wheat germribcsomes, whereas ricin A-chain, gelonin and tritin had little or no effect, even at much higherconcentrations. On the other hand, Harley and Beevers (1982) observed that ricin inhibited

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Battelli et al.—R ibosome-Inactivating Proteins and Plant R ibosomes 887

ribosomes from castor bean as well as from other plants. This difference could be due todifferent experimental conditions, amongst which there is the use, as a source of solubleenzymes, of a wheat-germ supernatant, which contains tritin (Roberts and Stewart, 1979;Coleman and Roberts, 1982). We confirmed that wheat-germ supernatant had no inhibitoryeffect on ribosomes from wheat germ, whilst inhibiting those from rat liver (results notshown). We used rat-liver supernatant throughout our experiment to ensure uniformity, and toprevent adding any RIPs that could be present in plant supernatants.

It is noteworthy that the inhibitory effect of increasing concentrations of PAP-S onribosomes from rice cells levels off when protein synthesis is approximately 50% of control.Furthermore, ribosomes from two carrot cell lines were highly sensitive to dianthin 32 andPAP—S and, to a lesser extent, to MCI, whereas ribosomes from carrot leaves were muchmore resistant. In the case of ribosomes from leaves, it is possible that the preparations usedcontain chloroplast ribosomes, which could be resistant to RIPs, like ribosomes from rat-livermitochondria (Greco, Montanaro, Novello, Saccone, Sperti and Stirpe, 1974). All together,these results seem to suggest the presence of different ribosomal populations in the sameorganism.

The inhibitory effect of PAP-S on the growth of carrot cells is consistent with the toxicityof PAP to tobacco protoplast (Grasso, Jones and White, 1980), and could be due to theaction of the protein on ribosomes. However, the difference between the effects of PAP-S onribosomes from rice and carrot cells is not such as to account for the lack of toxicity of thisRIP to rice cells. This could be due to other factors, such as different uptake or destruction ofPAP-S.

Present and previous results indicate (i) that ribosomes from various plant species aredifferent, and (ii) that RIPs from various plants are also different in their effect on the sameplant ribosomes. Thus many, and possibly all plants may contain proteins which recognizeand inactivate ribosomes 'different' from their own. A species specificity could not bedemonstrated under our experimental conditions, in that RIPs did not inactivate ribosomesfrom all heterologous species examined. However, RIPs have a more marked effect on animalribosomes, which are separated by a wider evolutionary gap from plant ribosomes. Thediversity of plant ribosomes indicated by experiments with RIPs is consistent with previousobservations (Ciferri and Parisi, 1970), with the species specificity of rRNA transcription(Grummt, Roth and Paule, 1982), and with the differential breakdown of RNA's (Lavelle,Ostro and Giacomoni, 1982).

The nature of the change(s) caused by RIPs on ribosomes is still unknown, and thus it isdifficult to assign a biological role to these proteins. It was suggested (Coleman and Roberts,1982) that they may be essential enzymes, or a defence against parasites. It could beenvisaged also that they may inactivate ribosomes from their own plants, should they bealtered, for instance by age or defect.

RIPs have been found in the sap or latex of some plants, in one case (the latex of Huracrepitans, unpublished results) at a concentration of 1 -0 mg cm"3, which could damagesusceptible cells. It is conceivable that, if one attempted to establish a graft on a stockcontaining a high level of an RIP toxic to the cells of the scion, these would be killed. Thus,RIPs could be one of the still unknown factors responsible for graft incompatbility amongstplants.

A C K N O W L E D G E M E N T SWe thank Dr. P. Ziska for a generous gift of Viscum album toxin. This research wassupported by the Consiglio Nazionale delle Ricerche, Rome, (grant no. 81.00163.04, GruppoNazionale di Biologia Molecolare, Cellulare ed Evolutiva and contract no. 81.01469.96,

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Progetto Finalizzato 'Controllo della crescita neoplastica'), by the Italian Ministero dellaPubblica Istruzione and by the Pallotti's Legacy for Cancer Research.

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