Kinetin counteracts the myomycin-inhibitory effect on plastid differentiation in excised cucumber cotyledons

Biochem. Physiol. Pflanzen 175,322-332 (1980)

Kinetin Counteracts the Myomycin-Inhibitory Effect on Plastid Differentiation in Excised Cucumber Cotyledons MARIA P. FASULO Institute of Botany, University of Ferrara, Italy Key Term Index: kinetin, myomycin, plastid differentiation, cotyledons; Cucumis sativus.

Summary The possibility of a specific action of kinetin(KN) on plastidial biosynthetic events was considered by verifying the ability of this phytohormone to contrast the inhibitory effect exerted on plastid differentiation by the antibiotic myomycin (MM), a specific plastid protein synthesis inhibitor. The experiments were carried out on etiolated cotyledons of cucumber (Cucumis sativus L.) incubated for 20 h in the dark followed by 24 h in the light in the presence or absence of KN (100 flM), or MM (200 flg/ml) or the two combined substances. In the presence of MM, chlorophyll synthesis and etioplast to chloroplast transformation were strongly inhibited while in the presence of KN they both were markedly enhanced. Importantly, when KN was applied simultaneously with MM, the inhibitory influence of the antibiotic was distinctly counteracted or completely nullified by the hormone. The bct that KN dominates the MM-inhibitory effect indicates that the action of the two molecules may be localized at similar cellular levels. Since MM exerts its activity by stably interacting with 70 S ribosomes of the chloroplast, it is suggested that KN could also act specifically at the same level.

Introduction

That cytokinins regulate numerous growth and developmental processes in plants is a well-established fact (for a review of literaturo see SKOOG and ARlVISTRONG 1970). Among these processes the promoting action of these hormonal compounds on the differentiation of plastids and chlorophyll synthesis is particularly emphasized (SUGIURA 1963; STETLER and LAETSCH 1965; FLETCHER and MCCULLAGH 1971a and b; FLETCHER et al. 1973; HARVEY et al. 1974; FARINEAU and ROUSSAUX 1970, 1975; WOZNY and SZWEYKOWSKA 1975; ROUSSEAUX et al. 1976; F ARINEAU et al. 1978; see review PARTHIER 1979). The effect appears to be the consequence of the cytokinin-stimulating action on protein and nucleic acid synthesis (OSBORNE 1962; BURDETT and WAREING 1968; ATKIN and SAHAI SRIVASTAVA 1970; KNYPL and CHYLINSKA 1972, 1973, 1974; FLETCHER et al. 1973; KLÄMBT 1976, MAASS and KLÄMBT 1.977). With respect to protein synthesis in particular, although the information found in literature is ambiguous and conflicting, it may be deduced that cytokinins affect plastid differentiation by promoting the process which occurs at cytoplasmic level (WOZNY and SZWEYKOWSKA 1975; F ARINEAU et al. 1978). Indications, however, exist that the compounds can also specifically favour plastidial biosynthetic processes (KULAEVA and ROMANKO 1968; YAKUSHKINA and PUSHKINA 1971; WOZNY and SZWEYKOWSKA 1975; Abbreviations: KN, kinetin; MM, myomycin.

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MIKULOVICH et al. 1978). The latter possibility is indirectly analysed in this papear by verifying the ability of a cytokinin to contrast the negative effect induced on plastid differentiation by a specific plastid protein synthesis inhibitor. The experiments were carried out on etiolated cotyledons of cucumber exposed to light in the presence of kinetin (RN) and myomycin (MM) (FRENCH et al. 1973). This is a new bleaching antibiotic for Euglena gracilis studied in our laboratory ; its mutagenic action is related to a selective permanent inhibition of protein synthesis on the 70S ribosomes of the chloroplast (FASULO et al. 1976a and b, 1977, 1979; VANNINI et al. 1978). The sensitivity of cucumber cotyledons to the antibiotic was already assured in preliminary experiments, while that to kinetin is weIl documented (FLETCHER and MCCULLAGH 1971a and b, WOZNY and SZWEYKOWSKA 1975). Furthermore, cucumber cotyledons have the advantage of being easily cultivable in vitra, of presenting numerous plastids and of permitting rapid experimentation (FLETCHER and MCCULLAGH 1971 b; F ARINEAU and ROUSSAUX 1975). Material and Methods Seeds of cucumber (Cucurnis sativus L. cv. Marketer) were germinated in the dark at 24°C for six days in the presence or absence of 200 f-lgjml of myomycin sulphate1 ). The germination extent was evaluated and seedling morphology was examined. The cotyledons were then excised and care was taken to remove the hypocotyl hook completely. The detached cotyledons were weighed and placed in nine cm Petri dishes on filter paper moistened with five ml of test solutions. All treatments were performed in dirn green safelight. Twenty cotyledons were used in each experimental variant. Since the myomycin molecule is very stable at pR 5.0 to 6.5 (FRENcH et al. 1973), all treatments were carried out in a 25 mM phosphate buffer at pR 5.5. The following concentrations of test substances, chosen on the basis of preliminary experiments, were used: 25 mM phosphate buffer pR 5.5 - Control myomycin sulphate at 200 f-lgjml concentration kinetin at 100 f-lM conc. myomycin at 200 f-lgjml and kinetin at 100 f-lM concs. The dishes with cotyledons were kept in the dark at 24°C for the next 20 hand then moved into continuous fluorescent light (150 ft-c). After 24 h of illumination, ten uniform cotyledons for each treatment were immediately blotted with filter paper, weighed and their chlorophyll content determined essentially as outlined by FLETcHER et al. (1973). Net growth of the cotyledons was calculated by subtracting the original fresh weight from the final fr. wt. of the organs. The tests were carried out in triplicate and repeated two times. For electron microscopy, the material was prepared basically as described by WOZNY and SWEYKOWSKA (1975) and examined with a Jeol JEM-T7 electron microscope operating at 60 KV. a) b) c) d)

Results

At the concentration employed (200,ag/ml), myomycin was practically non-toxic to cucumber seeds whose germination extent was fuUy comparablewith the control. After six days of dark germination, the MM-treated seedlings were slightly sm aller whereas their gross morphology was not influenced. Under electron microscopic examination, 1) Myomycin sulphate was obtained by courtesy of Parke, Davis & Co., Detroit, Mich, U.S.A.

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Figs. la and b. Etiolated cucumber cotyledons after a six-day germination in the absence (a) or in the presence (b) of 200 Mlml myomycin. ,. Both treated and untreated cotyledons show normally featured etioplasts containing so rne starch grains and para-crystalline pro lamellar bodies. 24,500 x; 27,500 x.

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Figs.2 and 3. Effect of myomycin (MM, 200 MImi), kinetin (KN, 100/kM) and the two combined substances (MM, 200 Mlml + KN, 100/kM) on fresh weight increase (Fig. 2) and chlorophyll synthesis (Fig. 3) in etiolated cucumber cotyledons.

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the cotyledons of the MM-treated seedlings were indistinguishable from the corresponding wild-type. In particular, they exhibited typical etioplasts containing some starch grains and one or two large crystalline pro lamellar bodies (Fig. la, b). In the cotyledons the negative effects of MM became evident at the end of a 24 h light exposure. Fresh weight increment and chlorophyll content were markedly inhibited and the effect was particularly exalted in the cotyledons arising from MM-germinating seedlings (Figs. 2 and 3). In contrast, when the etiola,ted cotyledons were exposed to light in the presence of kinetin, thcre was a marked stimulation of fresh weight and chlorophyll accumulation and the promotory effect was also evident when kinetin was applied to the cotyledons of the MM-germinating seedlings. Furthermore, when myomycin and kinetin were simultaneously supplied to the cotyledons, the antibiotic inhibitory effect on growth and chlorophyll synthesis was not only counteracted 01' nullified by the hormonal compound, but the cotyledons were sometimes even large and richer in photosynthetic pigments than the control itself. After 24 h of illumination, electron microscopic observation revealed detached cotyledons of the control group with developing plastids having appreciable membrane equipment which consisted of numerous thylakoids disposed in parallel bands in the stroma and normally appressed to form some granal structures (Fig. 4). Rarely, in these organelles, loose prolamellar bodies were still detectable, while starch grains were generally absent. In the presence of MM, plastid differentiation was clearly retarded 01' blocked in the early stages; the effect was more pronounced in the cotyledons of the MM-treated seedlings. The organelles, generally smaIl in size, were characterized by considerable polymorphism and the occurrence of numerous starch grains. The lamellar system consisted of a reduced number of thylakoids radially protruding from persisting crystaIline prolamellar bodies (Fig. 5) or irregularly arranged in the stroma (Fig. 6). In the more organized plastids, some aggregates composed of a few compartments could be observed, while singly-arranged peripheral or internal thylakoids frequently occurred within poorly structured, amoeboid plastids whose volume was entirely occupied by

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large starch granules (Fig. 7). When the etiolated cotyledons were kept in the light in the presence of kinetin, etioplast to chloroplast transformation was markedly accelerated. Plastids, elongated in shape, were starchless and contained an organized lamellar system. The thylakoids were weIl formed and both the number and depth of gran al stacking increased. No prolamellar body was detectable in the plastids, which in general showed the normal aspect of the fully developed organelles (Fig. 8). The kinetin promotive effect was also evident in the cotyledons of the MM-treated seedlings which were then exposed to light in the absence of the antibiotic (Fig. 9). Finally, when the cotyledons were simultaneously treated with MM and RN, the antibiotic inhibitory effect was counteracted by the hormone. Plastids, in fact, tended to be elongated in shape and to assume a regular outline (Figs. 10-12). An appreciable amount of intern al membrane structures were differentiating, and the organelles were fully comparable with the controls (Fig. 11). Furthermore, in the cotyledons of the MMtreated seedlings, extremely elongated plastids containing secondary prolamellar bodies were frequently observed (Fig. 12). Discussion

Myomycin inhibits chlorophyll synthesis and plastid differentiation in etiolated excised cucumber cotyledons exposed to light, apparently without affecting the other cellular components. The concomitant prevention of the fresh weight increment may be interpreted as a secondary effect resulting from a conceivably minor photosynthetic activity. As already stated in Euglena gracilis cells (FASULO et al. 1976a and b, 1977, 1979; VANNINI et al. 1978), the MM -specific effect on the biochemical and structural evolution of the plastid is due to a selective inhibition of protein synthesis in the 70 S bacterial-type ribosomes of the organelle. The inhibitory influence of the antibiotic, in fact, is undetectable at the level of the prolamellar body or the primary thylakoids, whose elaboration seems to depend essentially upon cytoplasmic biosynthetic events (DELSENEY et al. 1972; ROUSSAUX et al. 1976). During pre-incubation in the dark,

Fig.4. Cucumber cotyledons from control experiment. Six-day·old cotyledons incubated 14 h in darkness and 24 h in light. Developing plastid in which numerous thylakoids and some gran al eonfigurations may be observed. The prolamellar bodies are completely dispersed and starch grains are absent. 19,000 x. Figs.5-7. Cucumber cotylcdons trwted with 200pg/mlmyomycin (MM). After a six day germination in the absence (Fig. 5) or in the presence (Figs. 6 and 7) of 200,ag/ml MM, the cotyledons were incubated with the antibiotic 14 h in darkness and 24 h in light. Fig.5. Odd-shaped plastids with persistent pro lamellar bodies, thylakoids radially arranged and prominent star eh grains. 18,000 x. Fig.6. A poorly structured plastid with a few thylakoids variously oriented in the stroma. Large starch deposits are present. 19,500 x . Fig.7. Amoeboid plastid with severallarge starch grains surrounded by single thylakoids or packages of membrane associations arranged concentrieally. 27,000 x. 23 Biochern. Physiol. Pflanzen, Bd. 175

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Figs.8 and 9. Cucumber cotyledons treated with 100 p,M kinetin. After a six day germination in the absence (Fig. 8) or in the presence (Fig. 9) of 200 p,g(ml myomycin, the cotyledons were incubated with the hormone 14 h in darkness and 24 h in light. Fig.8. Chloroplast having all the characteristics of a fuHy developed organelle. 15,500 x. Fig.9. Chloroplast in an advanced stage of evolution when compared with the organelles shown in Figs.6 and 7. 18,000 x.

however, MM could prevent the synthesis of some precursors of the thylakoids, a fact which is revealed in the light by a rapid inhibition of the lamellar system formation. On the contrary in the presence of kinetin, both chlorophyll synthesis and plastid differentiation are stimulated. The higher pigment content and the building of a well developed granal system may be attributed to the activation of light-dependent biosynthetic processes utilizing both dark-accumulated precursors, and newly light-formed materials (FLETCHER et al. 1973; HARVEY et al. 1974; FARINEAU and ROUSSAUX 1975; FARINEAU et al. 1978). Considerable experimental evidencc supports thc view that this effect is the consequence of the KN-promotion of transcriptional and translational processes occurring during the dark and light incubation periods (SHA wand MANOCHA 1965; BHATTACHARYYA and Roy 1969; LETHAM 1969; SHORT et al. 1974; MAASS and KLÄMBT 1977; MIKULOVICH et al. 1978). Whether only cytoplasmic or also plastidial events are im plicated in the process is still unclear. In our case, the fact that the MM-inhibitory effect may be counteracted by KN indicates that the site of action of the two molecules may be localized at similar cellular levels. In particular, since MM exerts its activity by stably interacting with plastidial 70 S ribosomes it may be supposed that KN also acts upon the same structures. Such a hypothesis is supported by re cent data demonstrating highly

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Figs.10-12. Cucumber cotyledons treated with 200 flgjml myomycin and 100 flM kinetin. After a six day germination in the absence (Fig. 11) or in the presence (Figs. 10 and 12) of 200 flgjml myomycin, the cotyledons were incubated with the two substances 14 h in darkness and 24 h in light. Fig.10. Developing plastid exhibiting a regular outline and an appreciable lamellar system. 21,000 x. Fig.11. Plastid in a stage of evolution fully comparable with the contro!. 15,000 x. Fig.12. A very elongated plastid in which some secondary prolamellar bodies are present. 28,000 x.

specific bin ding sites in plant ribosomes for cytokinins (BERRIDGE et al. 1970, 1972; Fox and ERION 1975, 1977). The bin ding seoms to have a functional significance because a positive correlation was demonstrated by BERRIDGE et al. (1970, 1972) between the extent of bin ding and the biological effect of various cytokinin analogues. These Authors believe that 80S are the major species of ribosomes binding kinetin, but they do not 23*

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exclude the possibility that to a lesser degree plastidial 70S ribosomes mayaiso be involved. The overcoming of the MM-illhibitory effect by KN verifies the latter event. In this way KN, by billdillg on plastidial 70 S ribosomes, could protect these structures from the attack of MM, or modify the pre-existing MM-binding site thus making it inactive. In addition, the KN-binding to ribosomes could activate all the translation machinery, a process which would justify the KN-promotion of plastid differentiation. KN could also exert its protective action towards MM by stimulating a new synthesis of plastidial rRNAs, a fact already demonstrated in Cucumis sativus (ROUSSAUX et al. 1976) with another synthetic cytokinin (6-benzylaminopurine). In this case, t he possibility of a specific partecipation of kinetin in biosynthet ic events taking place in the plastidial compartment would be reinforced. It is known, in fact, that plastidial rRNAs are transcribed by a system synthesized chiefly within the plastid itself (HEIZMANN 1974). Acknowledgements This work was partially reported at the annual meeting of Cytology of Societa Botanica Italiana (Siena, Muy 1979) and was support ed by a grant from ItaJian Research Co uncil (CNR). The author wish to thank Professor G. D.UL'OLIO (University of Ferram) for t he critical appraisal of the manuscript, and Dr. D. l\IAREs for t he technical assistance.

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Received November 20, 1979. Author's address: Dr. MARIA P. FASULO, Institute of Botany, University of Ferrara, 1-44100 Ferrara, Italy.