Ethylene regulation of gene expression in carrots

Proc..Natl Acad. Sci. USA Vol. 79, pp. 4060-4063, July 1982 Botany

Ethylene regulation of gene expression in carrots (Daucus/hormone action/mRNA/polyribosomes)

ROLF E. CHRISTOFFERSEN AND GEORGE G. LATIES Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024

Communicated by Harry Beevers, April 15, 1982

KCV35 mM MgCl2/25 mM EGTA/5 mM 2-mercaptoethanol). The homogenate was filtered through Miracloth and then centrifuged at 2,500 rpm (1,000 x g) in an HB-4 Sorvall rotor. The supernatant was made 2% (wt/vol) with Triton X-100 and then centrifuged at 10,000 rpm (16,300 X g) in an HB-4 rotor. The resulting supernatant was layered over a 5-ml cushion of 1.8 M sucrose/40 mM Tris-HCI, pH 9.0/30 mM MgCI2/5 mM EGTA/ 5 mM 2-mercaptoethanol and centrifuged in a Beckman type 50.1 rotor at 42,000 rpm (196,000 X g) for 4 hr at 4°C. The resulting polyribosome pellet was resuspended in 500 pJ of resuspension buffer (40 mM Tris-HCI, pH 8.5/200 mM KCI/30 mM MgCl2/5 mM EGTA/5 mM 2-mercaptoethanol) by using a small Potter-Elvehjem homogenizer with aTeflon pestle. The resuspended polyribosomes (100 ,u1) were layered over a 15-50% (wt/vol) sucrose gradient made in 40 mM Tris-HCI, pH 8.5/20 mM KCV10 mM MgCl2. Polysomes were centrifuged at 50,000 rpm (337,000 X g) for 60 min in an SW 60 Beckman rotor at 4°C. The polyribosome profiles were analyzed with an ISCO gradient fractionator and UV absorbance monitor. Isolation ofPolysomal Poly(A)+RNA. RNA was isolated from polyribosomes as prepared above by resuspending the 42,000rpm pellet in 1 ml of 50 mM Tris-HCI, pH 7.5/10 mM EDTA/ 0.5% NaDodSO4; 500 ,ug of proteinase K (Beckman) was added to the suspension, and the mixture was incubated for 30 min at room temperature. Subsequently, RNA was precipitated by adding 0.1 vol of 3 M Na acetate (pH 6.0) and 2 vol of cold (-20°C) ethanol. Poly(A)+RNA was purified from the total polysomal RNA by two cycles of oligo(dT)-cellulose affinity chromatography (11). The RNA was assumed to have A2m = 1 at 40 ,ug/ml. In Vitro Translation. The reticulocyte lysate system of Pelham and Jackson (12) was used for in vitro translation of 0.5 jig of poly(A)+RNA in the presence of 50 ,uCi of [3S]methionine (1,000 Ci/mmol; 1 Ci = 3;7 X 1010°becquerels; New England Nuclear) in a total volume of 25 jl. Electrophoresis of in Vitro Labeled Translation Products. Aliquots of translation products containing equal quantities of trichloroacetic acid precipitable [3S]methionine were lyophilized and resuspended in NaDodSO4 sample buffer (13). Onedimensional electrophoresis was on 10-15% gradient acrylamide gels containing 1% NaDodSO4 (13). After fixation and staining, the gels were infiltrated with 2,5-diphenyloxazole, dried, and exposed to Kodak XR-5 film at -70°C (14). Twodimensional electrophoresis was performed as described by O'Farrell (15), except that aliquots of lyophilized translation products were resuspended in O'Farrell's lysis buffer containing 0.2% NaDodSO4 (16).

ABSTRACT The respiration ofcarrot (Daucus carota L.) roots is stimulated by ethylene. Polyribosomes were shown to proliferate concomitantly with the increase in respiration, and the ex*tent of polyribosome augmentation was closely correlated to the amount of respiratory stimulation. In addition to the increase in quantity, ethylene caused a 2-fold increase in the average polyribosome size, suggesting tighter pacldng of ribosomes on RNA. In vitro translation ofcarrot polyadenylylated RNA with the rabbit reticulocyte lysate system followed by electrophoresis of the resulting translation products showed that ethylene treatment results in the appearance of new mRNAs.

Although the involvement of ethylene in many aspects of plant growth and development is well documented, the mode of action is a relatively neglected area of study (1). The available evidence suggests that ethylene first binds to a metalloprotein (2), but subsequent events that lead to the observed phenomena are unknown. Many studies have demonstrated increases in specific enzyme activities in response to ethylene, and in some cases this increase is prevented by cycloheximide or actinomycin D (3-5). Whereas the foregoing implies that ethylene acts through the regulation of differential gene expression, this evidence is indirect and circumstantial. Carrot (Daucus carota L.) roots respond to ethylene in a manner that resembles the response of many storage organs-i.e., by an increase in respiration (6), an increase in phenylalanine ammonia-lyase activity (5), and a synergistic enhancement ofthe ethylene effect by pure 02(6, 7). A brief report (8) has indicated that ethylene enhances mRNA synthesis in potato tubers, several new polypeptides appearing among the mRNA in vitro translation products. In this paper we report the effect of ethylene on polysome profiles and mRNA populations in carrot roots. Our observations support the proposal that ethylene regulates gene expression. MATERIALS AND METHODS Plant Material. Carrots (D. carota L.), with their leaves intact, were purchased from local markets. Washed roots were placed in 4-liter glass jars and flushed for 24 hr at 20°C with a stream of water-saturated; air (50 mVmin) while carbon dioxide production was monitored with an infrared gas analyzer (Anarad, Santa Barbara, CA). Subsequently, experimental gases were introduced, and the measurement of CO2 evolution was continued. Polyribosome Isolation. The polyribosomes from carrot roots were prepared by the method of Jackson and Larkins (9), as modified by Goldberg et al..(10). Fresh tissue (15 g) was homogenized in ajuice extractor with 50 mnofcold (40C) extraction mixture (0.5. M.sucrose/200 mM Tris HCI, pH 9.0/400 mM

RESULTS Ethylene Effects on Respiration and Polyribosomes. Fig. 1 shows the respiration rate of carrot roots in response to ethylene given in air or in pure 02 for 24 hr. The carrots treated with C2H4/air displayed a 2-fold increase in rate, whereas those

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treated withC2H4/02 showed a 4-fold increase. The respiration rate was the same in air as in 02 in the absence of ethylene. After 24 hr of treatment with the designated gas mixtures, polyribosomes were prepared from the various tissues and analyzed on a sucrose gradient. Polyribosomes loaded on the- gradients represent equal amounts of tissue on a fresh weight. basis; results were consistent in four repetitions of the same experiment. The initial centrifugation at 42,000 rpm recovered essentially all the polyribosomes in the homogenate; longer times of centrifugation do not increase the polyribosome fraction but do increase the ribosomal subunit and monosome. fractions (R. Boyd, personal communication). Whereas oxygen alone was without effect, C2H4 caused a 1.7-fold polyribosome increase in air and a 4.9-fold increase in oxygen (Fig. 2). Polysomal Poly(A)"RNA Isolation and in Vitro Translation. Table 1 shows the amount of extracted polysomal total RNA and poly(A)+RNA from 02- and C2H4/02-treated carrot roots. The increase in total RNA due to ethylene is 4.8-fold. The foregoing value matches that obtained from the area under the polyribosome curve of the UV absorbance profile (Fig. 2). Polysomal poly(A)+RNA, in turn, increased 2.2-fold over the control. The polysomal poly(A)+RNA stimulated ['S]methionine incorporation 9- to 12-fold over the background incorporation of the rabbit reticulocyte lysate (results not shown). One-Dimensional Electrophoresis of Ethylene-Induced in Vitro Translation Products. Treatment of carrots with 02 alone had no effect on respiration, polyribosome content, or the oneand two-dimensional electrophoretic patterns of in vitro translation products (Fig. 3). Accordingly, we limited further studies to an examination of the effect of ethylene in 02. At least four predominant in vitro translation products appeared to be induced by ethylene (see arrowheads in Fig. 3), with Mrs of 80,000, 41,000, 38,000, and 37,000. One of the bands (Mr) 23,000) present in the control tissue was lost following ethylene treatment. The expected number of in vitro translation products was considerably greater (17) than the resolving capability of the one-dimensional electrophoretic system, at most 50 polypeptides. Accordingly, a two-dimensional electropho-

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polypeptides. Two-Dimensional Electrophoresis of Ethylene-Induced in Vitro Translation Products. The in vitro translation products of poly(A)+RNA isolated from 02- and C2H4/02-treated carrots were electrophoretically separated on a two-dimensional system that separates polypeptides on the basis of charge in the first dimension and molecular weight in the second dimension. The resulting fluorographs of the dried gels showed a complex pattern of in vitro label incorporation (Fig. 4). At least 500 individual polypeptides synthesized in vitro were detected by the two-dimensional system. The C2H4/02 treatment altered the Table 1. Yield of polysomal total RNA and poly(A)+RNA from carrot roots treated with 02 or C2H402 Yield, Ag/g fresh weight Poly(A)+RNA Polysomal total RNA Treatment 0.5 14.7 02 1.1 71.5 C2H4/02

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Proc. Nad Acad. Sci. USA 79 (1982)

Botany: Christoffersen and Laties

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FIG. 3. Fluorograph of electrophoretically separated in vitro translation products of carrot root poly(A)+RNA. Lanes: 1, endogenous products of reticulocyte lysate; 2, products of poly(A)+RNA from roots treated 24 hr with C2H4/02 at 10 1.l of ethylene per liter; 3, products of poly(A)+RNA from roots treated with 02 for 24 hr. Molecular weight size markers are shown x 10-3.

synthesis considerably. Examples of ethylene-induced in vitro translation products are enclosed in circles, whereas polypeptides lost in response to ethylene are enclosed in boxes (Fig. 4). pattern of in vitro protein

DISCUSSION The increase in respiration evoked by ethylene in carrots (Fig. 1) is a feature common to many bulky storage organs and is reminiscent ofthe ripening climacteric in fruit (6, 18). Slices of storage organs "aged" in solution also undergo a similar respiratory transition (19). It has been established that, during the washing period, slices show an increase in polysomes and a change in the in vitro translation products made from their poly(A)'RNA (20, 21). Furthermore, inhibitors of RNA or protein synthesis prevent the occurrence of the increase in respiration in slices (22). We have shown herein that ethylene evokes a similar increase in polysome content and the appearance of new mRNA species in intact organs (Figs. 2 and 3). The presentation of ethylene in pure has been observed to enhance the respiration of storage organs over that obtained with ethylene in air (6, 7). Thus, we can readily vary the intensity of the ethylene-evoked respiration increase simply by varying the oxygen tension in conjunction with ethylene treatment. The corresponding polyribosome profiles show that there exists a close relationship between the extent of the respiratory stimulation and the amount of polyribosome proliferation (Figs. 1 and 2). Various volatile compounds stimulate the respiration of storage organs much as does ethylene (23, 24). In every case so far examined by us, an increase in polyribosomes was coextensive with respiratory stimulation, independent of the evoking agent (i.e., cyanide, M. Tucker; ethanol, H. R. Highkin; personal communications). Respiration well may be responding to an energy demand created by either the act of protein synthesis per se or by the activity of newly synthesized enzymes. 02

FIG. 4. Fluorographs of two-dimensional electrophoretic separation of in vitro translation products from carrot roots. The poly(A)+RNA was prepared from roots treated with 02 for 24 hr (A) or with C2H4/ 02 (10 id/liter) for 24 hr (B). Circles, C2HA-induced products; boxes, products lost in response to C214.

Whereas the average polysome size changes upon ethylene treatment, from pentamer to decamer (Fig. 2), the poly(A)+RNA from polysomes of ethylene-treated tissue does not code for

longer translation products (Fig. 3). Furthermore, when the size distribution of poly(A)+RNA populations from control and ethylene-treated tissue was directly compared by methylmercury agarose gel electrophoresis, no significant size difference was noted (data not shown). This suggests that the ratio of ribosome to mRNA in polysomes has increased in response to ethylene. Supporting this hypothesis is the fact that, whereas both polysomal total RNA and polysomal poly(A)+RNA increase, the fraction of polysomal RNA that is poly(A)+RNA decreases by a factor of 2-i.e., from 3% to 1.5% (Table 1). This correlates well with the. 2-fold increase in the major polysome size class observed on the analytical sucrose gradients (Fig. 2). The packing ratio- of ribosomes on mRNA would be expected to increase if peptide chain elongation were inhibited or slowed while initiation continued unimpeded (25). It is unlikely that this is the case with ethylene because the protein content of carrots increases 40% after 48 hr of treatment with C2H4/air (5). Alternatively, the observed ethylene-induced increase in polysome packing could be the result of increased rates of peptide chain initiation. Further experiments are necessary to determine if the high molecular weight polysomes in ethylene-

Botany: Christoffersen and Laties treated tissue represent only newly induced mRNAs or whether both the new and constitutive mRNAs manifest higher ribosome packing ratios in response to ethylene treatment. Ethylene treatment induces a considerable change in the relative concentration of a number of mRNAs in carrot root (Figs. 3 and 4). One of the induced in vitro translation products has a molecular weight of 80,000. This is the reported molecular weight of the subunit of phenylalanine ammonia-lyase (26). Chalutz et al. (27) have shown that ethylene treatment of carrots results in the accumulation of isocoumarin and an increase in this enzymic activity that is blocked by actinomycin D and cycloheximide, respectively (5). We consider it likely that the observed Mr 80,000 ethylene-induced in vitro translation product represents the mRNA for phenylalanine ammonia-lyase, but this assumption must be confirmed by immunoprecipitation of the translation product with specific antiserum or by a peptide digest pattern. The functions of the other ethylene-induced and ethylene-removed mRNAs are completely unknown. In summary, we propose that ethylene acts, directly or indirectly, on two levels of genetic regulation: (i) the translational machinery, as evidenced by the proliferation of larger polysomes and the implication of tighter packing of ribosomes on mRNA; and (ii) the expression of specific messages, as shown by changes in the levels of in vitro translation products. Whether these changes are due to transcriptional control or to selection and transport of nuclear transcripts is not known. After we submitted this manuscript, a report by Zurfluh and Guilfoyle (28) appeared showing that Ethephon, an ethyleneforming compound, increases the level of several mRNAs in soybean hypocotyls. We thank Dr. J. Kamalay for helpful advice on preparing polyriboThis work was supported by a grant from the U. S. Public Health Service to G.G.L.

somes.

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3. Rhodes, M. J. C. & Woolhorton, L. S. C. (1971) Phytochemistry 10, 1989-1997. 4. Hyodo, H. & Yang, S. F. (1971) Plant Physiot 47, 765-770. 5. Chalutz, E. (1973) Plant Physiol 51, 1033-1036. 6. Theologis, A. & Laties, G. G. (1982) Plant Physiol 69, 1031-1035. 7. Chin, C. & Frenkel, C. (1977) Plant Physiol 59, 515-518. 8. Theologis, A. & Giudice, L. C. (1979) Plant Physiol 63, S-132

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9. Jackson, A. 0. & Larkins, B. A. (1976) Plant Physiol 57, 5-10. 10. Goldberg, R. B., Hoschek, G., Kamalay, J. C. & Timberlake, W. E. (1978) Cell 14, 123-131. 11. Aviv, H. & Leder, P. (1972) Proc. Natl Acad. Sci. USA 69, 1408-1412. 12. Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, 247-256. 13. Laemmli, U. K. (1970) Nature (London) 227, 680-685.

14. Laskey, R. A. & Mills, A. D. (1975) Eur.J. Biochem. 56, 335-341. 15. O'Farrell, P. H. (1975) J. Biol Chem. 250, 4007-4021. 16. Brandhorst, B. P. (1976) Dev. Biol 52, 310-317. 17. Kamalay, J. C. & Goldberg, R. B. (1980) Cell 19, 935-946. 18. Biale, J. B. & Young, R. E. (1981) in Recent Advances in the Biochemistry of Fruits and Vegetables, eds. Friend, J. & Rhodes, M. J. C. (Academic, London), pp. 1-39. 19. Laties, G. G. (1978) in Biochemistry of Wounded Plant Tissues, ed. Kahl, G. (de Gruyter, Berlin), pp. 421-466. 20. Kahl, G. (1978) in Biochemistry of Wounded Plant Tissues, ed. Kahl, G. (de Gruyter, Berlin), pp. 347-390. 21. Ishizuka, M., Sato, T., Watanabe, A. & Imaseki, H. (1981) Plant Physiol 68, 154-157. 22. Click, R. C. & Hackett, D. P. (1963) Proc. Natl Acad. Sci. USA 50, 243-250. 23. Rychter, A., James, H. W., Chin, C. & Frenkel, C. (1979) Plant Physiol 64, 108-111. 24. Solomos, T. & Laties, G. G. (1975) Plant Physiol 55, 73-78. 25. Weeks, D. P. (1981) in The Biochemistry of Plants, ed. Marcus, A. (Academic, New York), Vol. 6, pp. 491-529. 26. Tanaka, Y. & Uritani, I. (1977) J. Biochem. 81, 963-970.

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