Changes in Cotyledon Mrna During Ethylene Inhibition of Floral Induction in Pharbitis Nil Strain Violet

Plant Physiol. (1987) 84, 545-548 0032-0889/87/84/0545/04/$O 1.00/0

Changes in Cotyledon mRNA during Ethylene Inhibition of Floral Induction in Pharbitis nil Strain Violet

Received for publication October 21, 1986 and in revised form December 26, 1986

MICHAEL LAY-YEE', RoY M. SACHS, AND MICHAEL S. REID* Department ofEnvironmental Horticulture, University ofCalifornia, Davis, California 95616

ABSTRACT during a 16 h inductive dark period inhibited flowering (12, 13). Floral induction in seedlings of Pharbitis nil strain with one Using a static gas treatment system, Suge (13) found that 100 II/ Violet, L ethylene inhibited flowering of Pharbitis seedlings when ap- cotyledon removed, was manipulated by applying various ethylene treat- plied in the latter halfofa 16 h dark period but not when applied ments to the remaining cotyledon during a 16 hour inductive dark period. for 8 h from the beginning of the dark period. Some caution is Exposure of cotyledons to ethylene (100 microliters per liter) for 4 hours in at different times during the dark period inhibited flowering to some required interpreting these findings because static treatments extent, with inhibition being greater towards the end of the dark period. could result in elevated CO2 levels, which can inhibit ethylene RNA from cotyledons given a 16 hour dark period (induced) or exposed action (3). to 100 microliters per liter ethylene throughout the dark period, which The mechanism by which ethylene inhibits floral induction in completely inhibited flowering, was examined. The poly(A)+RNA was SDPs is unknown. Suge (12) concluded from his studies on P. translated in vitro using a wheat germ system, and the resulting transla- nil that ethylene had no effect on the translocation of a 'floral tion products were analyzed by two-dimensional sodium dodecyl sulfate- stimulus' from cotyledons to the apex, and that ethylene-treated polyacrylamide gel electrophoresis. There were substantial qualitative cotyledons did not transfer any flower inhibiting entity. He and quantitative differences between the poly(A)'RNA extracted from suggested that ethylene was inhibiting the induction process(es) induced cotyledons and that from those exposed to ethylene throughout in the cotyledons. In a later paper, Suge (13) reported that the the dark period. Some of these changes are similar to those observed endogenous gibberellin (GA) content decreased by approxi- when flowering was inhibited by photoperiodic treatments (M Lay-Yee, mately 30% in ethylene-treated plants, and that GA3, GA7, or RM MS Reid 1987 Planta. In The of these kinetin applied at the start of ethylene treatment, partially re- Sachs, press). significance stored flowering in ethylene treated plants. He concluded, never- findings to our understanding of the molecular control of flower induction theless, that the mechanism of flower inhibition by ethylene was is discussed. unlikely to be due to effects on the biosynthesis or action of endogenous GAs. He suggested that the effect of applied GAs and kinetin on ethylene-induced inhibition might be due to indirect effects of these chemicals. We have recently demonstrated qualitative and quantitative changes in mRNA populations following flower induction in P. nil strain Violet (9). Abeles (1) suggested that in Xanthium Ethylene has been reported to affect the flowering behavior of ethylene may interfere in some way with the 'orderly' production a number of plants. Cooper and Reece (8) found that a 6 h of RNA required for the flowering process. Ethylene has been treatment with 1000 ,l/L ethylene induced 100% flowering in found to affect gene expression in a number of systems. For pineapple. Ethephon (2-chloroethylphosphonic acid), an ethyl- example, ethylene enhanced RNA and protein synthesis in bean ene releasing compound, was found to induce flowering in abscission zone explants prior to abscission (2). Ethephon was mango (4, 5) and treatment of the SD2 plant Plumbago indica found to increase the level of several mRNAs in intact soybean L. with ethephon was also found to promote flowering (10). hypocotyls (16), and Christoffersen and Laties (6) found that the In contrast, the flowering of many of the classical SDPs is rise in respiration of ethylene treated carrot roots was associated inhibited by ethylene. In Xanthium pennsylvanicum, flower for- with an increase in polyribosome number and size, and the mation was fully suppressed by 10 and 100 Ml/L ethylene applied appearance of new mRNAs. during a 16 h inductive dark period (1). Tjia et al. (14) found The aim ofthe work described here was to study the sensitivity that flower bud production of Chrysanthemum morifolium 'In- of P. nil seedlings to ethylene at different times during a 16 h dianapolis White,' was inhibited by continuous exposure to 1 to dark period using a flow-through gas treatment system, and to 4 yd/L ethylene in short days. Ethylene was more effective when investigate the effects of ethylene on cotyledon gene expression given during the dark period than when applied during the light during an inductive dark period. It was hoped that in addition period. Cockshull and Horridge (7) reported that 100 or 1000 to clarifying the mode of action of ethylene in inhibiting flow- ,ul/L ethephon delayed flower initiation in C. morifolium 'Polaris' ering, the results would serve as another probe for investigations when applied as a foliar spray at the onset ofshort days. Ethylene of the photoperiodic control of flowering at the molecular level (100 ,ul/L) applied to seedlings of Pharbitis nil strain Violet (9).

' Recipient ofa Fulbright travel grant and a New Zealand Department of Scientific and Industrial Research study award. Present address: Di- MATERIALS AND METHODS vision of Horticulture and Processing, Department of Scientific and Plant Material. Seedlings of Pharbitis nil can be induced to Industrial Research, Private Bag, Auckland, New Zealand. flower at 23°C with a single 16 h dark period. Flowering can be 2Abbreviation: SD, short day; E, ethylene. inhibited by giving ethylene (100 ul/L or more) during the 545 546 LAY-YF,E ET AL. Plant Physiol. Vol. 84, 1987 inductive dark period (12). The cotyledons have been shown to be the site of perception of the photoperiod (1 1), and also the site of ethylene action in the inhibition of flowering (12). f--z The seeds were treated with concentrated H2SO4 for 30 min, -j rinsed well, and soaked for 7 h in aerated deionized H20. The 0. seed was then germinated on moist filter paper in the dark at 0- 27C. When the emerging radical was approximately 5 mm in cn length the seedlings were planted in 1:1:1 peat:sand:bark in 6 x 4 x 5.5 cm plastic pots. The developing seedlings were grown -LJ I-jw under continuous light (Norelco metal halide R, 400 W, 250 ;E U- m-2 s-') at 27C and watered daily with modified Hoagland nutrient solution. Seven d after the acid treatment the cotyledons were expanded and uniform plants were selected for treatment. wm One cotyledon was removed from each plant and the remaining z in a cotyledon enclosed black Plexiglas chamber, thus isolating tJ it from the apex and the rest of the plant, which were kept in continuous light (Phillips VHO/EW 185 W/ 1500 mamp, 30 ,uE m-2 s-' at plant level). Each Plexiglas chamber was equipped for flow-through gas treatment. Ethylene Sensitivity of Pharbitis Seedlings. To study the sensitivity of the plant to ethylene at different times during the 16 HIOURS OF ETHYLENE TREATMENT h dark period, the enclosed cotyledons were subjected to ethylene FIG. 1. Effect of ethylene treatment at different times during the 16 (100 ,ul/L) continuously, or for 4 h starting 0, 4, 8, or 12 h after h dark period on flowering ofP. nil. Bars with no letters in common are the start of the dark periods. The cotyledons were maintained in significantly different (P = 0.05). the dark in ethylene-free air when not being treated with ethylene. The temperature was maintained at 23 ± 2C during the 16 h Effect of Ethylene on Cotyledon Poly(A)'RNA. RNA yields treatment period, following which plants were returned to con- were determined spectrophotometrically by UV absorption. The tinuous light (Norelco metal halide R, 400 W, 250 uE m-2 s-', yields ofpoly(A)+RNA were determined from the pooled samples 400-700 nm), at 27C. The flowering response was determined comprising fractions, eluted from the oligo-dT-cellulose column, as the average number offlower buds per plant and was evaluated with highest A at 260 nm. From cotyledons subjected to SD and after 15 d. Each treatment was applied to three replicates of five E, the yields of total RNA (0.82 and 1.00 mg/g fresh weight, plants each. respectively), and poly(A)+RNA (10.4 and 12.4 ,g/g fresh weight, Effects of Ethylene on Cotyledon Poly(A)'RNA. To study the respectively), were similar. effects of ethylene given during a 16 h inductive dark period on Poly(A)+RNA populations from SD and E cotyledons were the mRNA population of Pharbitis cotyledons, seedlings, pre- first compared by one-dimensional gel electrophoresis of the pared as above, were given a 16 h dark period throughout which corresponding translation products. Without the addition of they were treated with either air (SD) or 100 ,l/L ethylene (E). exogenous poly(A)+RNA the wheat germ system produced no Following treatment, the cotyledons were harvested, frozen in detectable radiolabeled polypeptides (Fig. 2). When Pharbitis liquid N2, and stored at -80C pending RNA extraction. cotyledon poly(A)+RNA was added a number of radiolabeled Poly(A)+RNA in the RNA extracted from the frozen cotyledons bands were observed. Several differences were detected between was isolated on an oligo-DT cellulose affinity column and trans- the SD and E treatments, but this one-dimensional analysis lated in a wheat germ in vitro system using [35S]methionine to provided relatively low resolution of the translation products, label the polypeptides. The radioactively labeled polypeptides separating only approximately 35 radiolabeled bands. were separated by one- and two-dimensional PAGE, and visu- Two-dimensional gel electrophoresis of translation products alized by fluorography. The details of these techniques are de- from the treatments resulted in the separation of over 300 scribed elsewhere (9). Five plants from each treatment were polypeptides with mol wt ranging from 20 to 90 kD and pls from grown in continuous light (Norelco metal halide R, 400 W, 250 4.2 to 7.6 (Fig. 3). Gels shown in Figure 3 represent translation ,uE m-2 s-', 400-700 nm) at 27°C for 15 d after treatment to products from one set oftreated plants. The differences indicated check their flowering response. in the polypeptide patterns were found in all replications. Ethyl- Significance of Polypeptide Pattern. The experiment outlined ene treatment resulted in both qualitative and quantitative above (plant treatment, RNA extraction, in vitro translation, changes in the in vitro translation products of cotyledon two-dimensional gel electrophoresis, and fluorography) was rep- poly(A)+RNA. Some translation products were found in ethylene licated four times. The RNA from each set of treated plants was treated cotyledons that were either not present (spots 1 and 4) or translated at the same time. Aliquots of the resulting polypep- much reduced (spots 2 and 3) in control cotyledons. Other tides, from the two treatments, were simultaneously subjected to translation products were reduced in ethylene treated cotyledons two-dimensional gel electrophoresis so that separation was car- (spots 5, 6, and 7). ried out under identical conditions, thus producing gels that were Computer analysis ofthe fluorographs ofthe two-dimensional directly comparable. gels (13, 14), confirmed the differences described above (data not shown). The isoelectric points and mol wt of the significant in vitro translation products described above are shown in Table I. RESULTS Ethylene Sensitivity of Pharbitis Seedlings. Ethylene given DISCUSSION throughout the 16 h dark period completely inhibited flowering ofPharbitis seedlings (Fig. 1). Each ofthe 4 h ethylene treatments As reported by Suge (12), treatment of cotyledons ofPharbitis inhibited flowering (P = 0.05) to some extent. Inhibition was nil strain Violet with 100 ul/L ethylene throughout the inductive greater when the ethylene treatment was applied in the middle 16 h dark period completely inhibited flowering. Even when of the dark period (from 8-12 h) than when it was applied at cotyledons were treated only for 4 h at different times during the other times. dark period, ethylene strongly inhibited flowering. Suge (13) mRNA CHANGES AND INHIBITION OF FLOWERING BY ETHYLENE 547 1 2 3 found that treatment of Pharbitis seedlings with 100 41/L ethylene during the latter half of the dark period inhibited flowering; MW however, he also reported that ethylene given during the first 8 X10-3 h of a 16 h dark period did not inhibit flowering. This latter finding is not consistent with the results reported here. In the experiments described by Suge, whole seedlings were treated with ethylene in a static treatment system. In the present experiment - 92.5 only the cotyledons were treated, and possible CO2 accumulation which could inhibit ethylene action (3) was avoided by the use of a flow-through treatment system. These differences in experi- mental procedure may account for the inconsistency described - 66.2 above.

Table I. Characterization ofsome ofthe in Vitro Translation Products (Fig. 3) ofPharbitis Cotyledon Poly(A)RNA which Changed Markedly as a Result ofTreating the Cotyledons with 100 #1/L Ethylene - 45.0 Throughout the 16 h Dark Period In Vitro Isoelectric Translation Product Molwt Point kD 1 44 5.1 2 39 5.75 3 37 6.1 - 31.0 4 42 6.5 5 54 7.25 6 32 6.55 7 28 6.6

Table II. Yields ofTotal RNA and Poly(A)+RNA (determined by UV absorption)from P. nil Cotyledons Subjected to a 16 h Inductive Dark - 21.5 Period (SD) or to 100 ppm Ethylene Throughout a 16 h Dark Period (E) Poly(A)+RNA yields were determined from the pooled samples com- SD E 0 prised of fractions, eluted from the oligo-dT-column, with highest ab- FIG. 2. Fluorograph of [35S]methionine-labeled in vitro translation sorbances at 260 nm. Analysis of variance of the data showed no products obtained from poly(A)+RNA from Pharbitis cotyledons and significant differences between the treatment means (P = 0.05). resolved by SDS-PAGE. Cotyledons were subjected to a 16 h dark period Total RNA (lane 1) or 100 ld/L ethylene throughout the dark period (lane 2). In f Coatlmednts Poly(A)+RNA Poly(A)+RNA vitro translation was performed in a wheat germ cell-free protein-synthe- mg/gfresh wt wt % oftotal RNA sizing system. Lane 3 shows the endogenous level ofprotein synthesis by ug/ggfresh the wheat germ system in the absence of added Pharbitis cotyledon SD 0.819 10.38 1.27 poly(A)+RNA. E 1.007 12.37 1.24 pH4.2 _No...... pHZ6 pH4.2 IEF pH 7.6 cn A B MW cn) x10-3 -92.5 CD rr -A& I

4 4~~~~~~~~~~~~~~~~~~~~~~~~~4.

-31.0

- 9- Stj|=2**,}{!-215{r @ FIG. 3. Fluorograph of [35S]methionine-labeled in vitro translation products obtained from poly(A)+RNA from Pharbitis cotyledons and resolved by two-dimensional gel electrophoresis. Cotyledons were subjected to a 16 h dark period (A) or 100 Ml/L ethylene throughout the dark period (B). In vitro translation was performed in a wheat germ cell-free protein-synthesizing system. Spots considered significant are circled and numbered. See also Table I. 548 LAY-YEE ET AL. Plant Physiol. Vol. 84, 1987 In Pharbitis the time of maximum sensitivity to a night-break California, Davis) for kindly allowing us to use his laboratory facilities for much of is 8 to 10 h after the end ofa continuous photoperiod (15). Since this research, and Drs. B. Bonner and J. Harada (University of California, Davis) ethylene-induced inhibition was maximal at this same time (Fig. for their helpful advice during the preparation ofthis manuscript. 1), it is possible that ethylene and red light inhibit flowering by affecting similar processes. LITERATURE CITED Although levels of total RNA and poly(A)+RNA from cotyle- 1. ABELES FB 1967 Inhibition of flowering in Xanthium pensylvanicum WalIn. dons of SD and E plants were not significantly different (Table by ethylene. Plant Physiol 42: 608-609 II), poly(A)+RNA populations were strongly affected by the 2. ABELES FB, RE HOLM 1966 Enhancement ofRNA synthesis, protein synthesis, and abscission by ethylene. Plant Physiol 41: 1337-1342 ethylene treatment. Abeles (1) suggested that ethylene may in- 3. BURG SP, EA BURG 1967 Molecular requirements for the biological activity of hibit flowering by interfering with the pattern ofgene expression ethylene. Plant Physiol 42: 144-152 required for floral induction. The present findings support this 4. CHACKO EK, RR KOHLI, GS RANDHAWA 1974a Investigations of the use of proposal and demonstrate that, at the mRNA level, ethylene can (2-chloroethyl)phosphonic acid (Ethephon, CEPA) for the control ofbiennial bearing in mango. Sci Hortic 2: 389-398 simultaneously enhance the expression of certain genes and 5. CHACKO EK, RR KOHLI, R DORE SWAMY, GS RANDHAWA 1974b Effect of(2- inhibit the expression of others. chloroethyl)phosphonic acid on flower induction in juvenile mango (Man- Because ofthe relatively large number of messages affected by gifera indica) seedlings. Physiol Plant 32: 188-190 the ethylene treatment, identification of any which may be 6. CHRISTOFFERSEN RE, GC LATIES 1982 Ethylene regulation of gene expression in carrots. Proc Natl Acad Sci USA 79: 4060-4063 specifically linked to the regulation of flowering was difficult. 7. COCKSHULL KE, JS HORRIDGE 1978 2-chloroethylphosphonic acid and flower One message (spot 7) coding for a 28 kD polypeptide with a pl- initiation by Chrysanthemum morifolium Ramat. in short days and in long of 6.6 was found to be at reduced levels in E cotyledons relative days. J Hortic Sci 53: 85-90 to SD cotyledons (Fig. 3). In previous work (9), a similar 8. COOPER WC, PC REECE 1942 Induced flowering of pineapples under Florida conditions. Proc Florida State Hortic Soc 54: 132-138 poly(A)+RNA, was found to be at a reduced level in cotyledons 9. LAY-YEE M, RM SACHS, MS REID 1987 Changes in cotyledon mRNA during of plants in which floral induction was inhibited by a red night- floral induction of Pharbitis nil strain Violet. Planta. In press break. The correlation between the inhibition offloral induction 10. NITSCH C, JP NITSCH 1969 Floral induction in a short-day plant Plumbago and reduced levels ofthis poly(A)+RNA species, in both ethylene indica L. by 2-chloroethanephosphonic acid. Plant Physiol 44: 1747-1748 1 1. OGAWA Y, RW KING 1979 Indirect action of benzyladenine and other chem- and night-break treated cotyledons, suggest that the message may icals on flowering ofPharbitis nil Chois. Plant Physiol 63: 643-649 be involved in the induction process in P. nil. 12. SUGE H 1972 Inhibition of photoperiodic floral induction in Pharbitis nil by Recombinant DNA techniques are now being utilized to iso- ethylene. Plant Cell Physiol 13: 1031-1038 late cDNA clones to this message and others linked to floral 13. SUGE H 1974 Nature of the ethylene inhibition of flowering in Pharbitis nil. In Plant Growth Substances 1973. Hirokawa, Tokyo, pp 960-966 induction in Pharbitis cotyledons. These clones will enable fur- 14. TJIA BOS, MN ROGERS, DE HARTLEY 1969 Effects ofethylene on morphology ther investigation ofthe role of gene expression in the control of and flowering of Chrysanthemum morifolium Ramat. J Am Soc Hortic Sci flowering in this species. 94: 35-39 15. VINCE-PRUE D, J GRESSEL 1985 Pharbitis nil. In AH Halevy, ed, Handbook Acknowledgments-We wish to thank Dr. J. D. Anderson, Dr. W. P. Hruschka, of Flowering, Vol IV. CRC Press, Boca Raton, FL, pp 47-8 1 and Adele Robbins, United States Department of Agriculture, Beltsville, MD, for 16. ZURFLUH LL, TJ GUILFOYLE 1982 Auxin- and ethylene-induced changes in their expert guidance and for the use of their computer facilities in the analysis of the population of translatable messenger RNA in basal sections and intact two-dimensional gels. We would also like to thank Dr. A. Bennett (University of soybean hypocotyl. Plant Physiol 69: 338-340