The Ethylene-Regulated Expression of CS-ETR2 and CS-ERS Genes in Cucumber Plants and Their Possible Involvement with Sex Expression in Flowers

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1 Plant CellPhysiol. 4(5): (2000) JSPP 2000 The Ethylene-Regulated Expression of and Genes in Cucumber Plants and Their Possible Involvement with Sex Expression in Flowers Seiji Yamasaki, Nobuharu Fujii and Hideyuki Takahashi' Institute of Genetic Ecology, Tohoku University, 2-- Katahira, Aoba-ku, Sendai, Japan It has been reported that ethylene production by cucumber plants is strongly related to the sex expression of their flowers. It has also been shown that both CS-ACS2 gene expression and ethylene evolution are much greater in gynoecious cucumber plants than monoecious ones. To investigate the action mechanism of ethylene in the induction of femaleness of cucumber flowers, we isolated three ethylene-receptor-related genes,, and, from cucumber (Cucumis sativus L.) plants. Of these three genes, and mrna accumulated more substantially in the shoot apices of the gynoecious cucumber than those of the monoecious one. Their expression patterns correlated with the expression of the CS-ACS2 gene and with ethylene evolution in the shoot apices of the two types of cucumber plants. Accumulation of and mrna was significantly elevated by the application of Ethrel, an ethylene-releasing agent, to the shoot apices of monoecious cucumber plants. In contrast, the accumulation of their transcripts was lowered when aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene biosynthesis, was applied to the shoot apices of gynoecious cucumber plants. Thus, the expression of CS- ETR2 and is, at least in part, regulated by ethylene. The greater accumulation of and mrna in gynoecious cucumber plants may be due to the higher level of endogenous ethylene, which plays a role in the development of female flowers. Key words: Cucumis sativus Cucumber Ethylene Ethylene receptor Sex expression. There are three typical patterns of sex expression in cucumber plants, namely, monoecious (the most common type of sex expression), gynoecious, and hermaphroditic (Malepszy and Niemirowicz-Szczytt 99). Monoecious Abbreviations: ACC, -aminocyclopropane-l-carboxylate; AVG, aminoethoxyvinyl glycine; RT-PCR, reverse transcriptional polymerase chain reaction. The nucleotide sequences in this paper have been submitted to the DDBJ/GenBank DNA under accession numbers AB (), AB () and AB (). Corresponding author: , hideyuki@ige.tohoku.ac.jp; Fax, plants produce male flowers at the base of the main stem, then male and female flowers on the middle part, and finally female flowers at the top. The gynoecious type of cucumber plant produces only female flowers. All floral buds that differentiate at axes contain primordia of stamens and a pistil at the early stage of their development. They subsequently develop into either male or female flowers depending on the genotype and the environmental conditions under which the plants are grown (Atsmon and Galun 960, Takahashi et al. 983). The sex expression of cucumber plants is also influenced by plant hormones. Ethylene and auxin promote the formation of female flowers, whereas gibberellins promote the formation of male flowers (Galun 959, Peterson and Anhder 960, MacMurray and Miller 968, Iwahori et al. 970, Takahashi and Suge 980, 982, Saito and Takahashi 987). The enhancement of femaleness by auxin possibly occurs through the induction of ethylene biosynthesis (Takahashi and Jaffe 984, Trebitsh et al. 987). It is considered that ethylene acts more directly than gibberellins on sex expression of cucumber flowers (Yin and Quinn 995). Ethylene evolution is highly correlated with sex expression; gynoecious cucumber plants produce more ethylene than monoecious ones (George 97, Rudich et al. 972, Trebitsh et al. 987). Furthermore, inhibitors of ethylene biosynthesis or action suppress the development of female flowers and induce male ones (Beyer 976, Atsmon and Tabbak 979, Takahashi and Suge 980, Takahashi and Jaffe 984). These results suggest that ethylene is a regulator in determining sexuality of cucumber plants. The relationship of ethylene evolution to the femaleness of cucumber flowers was further demonstrated by Trebitsh et al. (997), who reported that the CS-ACSJ gene encoding -aminocyclopropane-l-carboxylate (ACC) synthase was auxin-inducible and that the CS-ACSG gene exists in gynoecious lines but not in monoecious lines. Kamachi et al. (997) also showed that the CS-ACS2 gene encoding ACC synthase was expressed during the development of female flowers in the shoot apices of gynoecious cucumber plants. These results suggest that CS-ACS/2 genes are closely linked to the female (F) locus that enhances the femaleness in cucumber. However, the action mechanism of ethylene on sex determination has not been fully elucidated. As has been done in other plant species (Bleecker et al. 988, Chang et al. 993, Ecker 995, Hua et al. 995), not only 608

2 Ethylene receptors and sex expression in cucumber 609 the level of ethylene production but also the tissue sensitivity to ethylene needs to be studied to elucidate the mechanism of ethylene action in cucumber plants. Recently, the application of molecular and genetic studies to Arabidopsis has allowed identification of the ethylene receptors (Chang et al. 993, Hua et al. 995, 998, Sakai et al. 998). The ethylene signal appears to be perceived by a family of ethylene receptors. Five members of the putative ethylene receptor gene family, ETR, ERS, ETR2, EIN4 and ERS2, have been cloned (Chang et al. 993, Hua et al. 995, 998, Sakai et al. 998). All of them confer dominant insensitivity to ethylene under a variety of physiological conditions. Analysis of loss-offunction mutants of four of these genes (ETR, ETR2, EIN4 and ERS2) has revealed that these proteins act redundantly and negatively regulate ethylene responses. Accordingly, the induction of the ethylene response is mediated through inactivation of these proteins (Hua and Meyerowitz 998). Double-mutant analysis has demonstrated that all five members of this gene family act upstream of all other signal transduction genes isolated by the triple response assay (Ecker 995, Hua et al. 998, Sakai et al. 998). A similar approach may be worthwhile for clarifying the signal transduction mechanism of ethylene in inducing femaleness in cucumber flowers. As yet, there have been no ethylene receptors isolated from cucumber plants and no evidence for any relationship between such ethylene receptors and sex expression in cucumber plants. To study the action mechanism of ethylene in the induction of femaleness in cucumber plants, we isolated three ethylene-receptor-related homologs from cucumber plants and examined their expression patterns in monoecious and gynoecious cucumbers. We also attempted to answer the question of whether or not the difference in ethylene production between the two types of cucumber plants is involved in the expression of the ethylene-receptor-related genes. Materials and Methods Plant materials Monoecious {Cucumis sativus L. cv. Otone No.l) and gynoecious {Cucumis sativus L. cv. Higan-fushinari) cucumber plants were used in this study. Characteristics of sex expression of these cucumber plants have been reported elsewhere (Takahashi and Suge 980, 983). In brief, most cucumber plants, including Otone No., prefer short-day conditions for the production of female flowers. However, Higan-fushinari becomes completely gynoecious under long-day conditions and produces male flowers on the lower nodes of the main stem under short-day conditions. Seeds of the two types of cucumber plants were germinated on wet filter paper in a Petri dish at 28 C in the dark for to 2 d. These seedlings were transferred to plastic pots (2-cm in diameter) containing a soil composite, Kureha-Engei-Baido (0.4 g N,.9 g P, 0.6 g K per kg, Kureha Chemical Co., Tokyo). Plants were grown under a 24-h photoperiod, with daylight being supplemented by fluorescent lamps in a greenhouse. Plants were adequately watered and supplied with fertilizer, 0.002% (v/v) Hyponex (Hyponex-Japan, Osaka). Isolation of total RNA To obtain RNAs from the shoot apices of cucumber plants at different stages of growth, the stages of growth were classified as follows. In the seedling stage (cotyledonary stage), the cotyledons were approximately 2-cm long. Plants with the third, fourth, fifth and eighth leaves, each expanded to a length of approximately 2 cm, were defined as being in the 3- stage, 4- stage, 5- stage and 8- stage, respectively. In this study, the term shoot apex means the apical shoot including immature leaves but not expanded leaves longer than 2 cm. Leaves, stems and roots below the expanded leaves of the plants were discarded. At different stages of growth, the shoot apices of cucumber plants were cut off with a razor blade, immediately frozen in liquid nitrogen, and stored at 80 C prior to the extraction of nucleic acids. When chemicals were applied, shoot apices were cut off 8 h and 4 d after the start of the treatment. Total RNA was extracted from those samples using ISOGEN (Nippongene, Tokyo), according to the manufacturer's instruction. Isolation of cdnas for ETR homologs To isolate ETR fragments, we synthesized the degenerate oligonucleotide primers homologous to the conserved regions of ETR. The degenerate oligonucleotides FER, 5'-G[A/C][A/G]GA[A/G]TGTGC[T/A]- TT[T/A/G]TGGATGCC-3' and RER, 5-TGCAT[A/T]GG[A/ T]GT[T/C]CTCAT[T/C]CCATGATTCAT-3' corresponded to the amino acid sequences of AEECALWMP and MNHEMRTP- MH, respectively. For RT-PCR (Reverse transcriptional polymerase chain reaction), cdna was synthesised from total RNA of the shoot apices of Otone No. at the 4- stage, using Moloney Murine Leukemia Virus (M-MLV) Reverse transcriptase RNaseH Minus (Toyobo Co. Ltd., Osaka) with random hexemar as primer. Partial cdna fragments designated, and were amplified by RT-PCR using FER and RER as primers. The products of PCR were analyzed by gel electrophoresis on 2.O /o agarose gel and recovered. The isolated partial cdna product was cloned into the pgem R -T Vector (Promega Inc., Madison) by the method of TA Cloning. To isolate full-length cdnas of, and, we screened lamda-zapii (Strategene, La Jolla) cdna libraries produced from cucumber hypocotyl segments. A nylon membrane (Nytran NY3N; Schleicher and Schuell, Dassel) duplicated library for plaque hybridization (Benton and Davis 977) was probed with fragments of,, and genes according to the instructions provided with the DIG RNA Labeling and Detection Kit (Boehringer Mannheim, Mannheim). DNA sequencing DNA sequencing was performed by the dideoxy sequencing method (Sanger et al. 977) using a Taq Dye Primer Cycle Sequencing Kit (Perkin-Elmer Japan Co. Ltd., Urayasu) and a DNA sequencer (model 377; Perkin-Elmer Japan Co. Ltd.). Genomic DNA gel blot analysis Genomic DNA was extracted from cucumber leaves by the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson 980). Two micrograms of DNA isolated from leaves of the two cucumber plants, Otone No. and Higan-fushinari, were separately digested with three kinds of restriction endonucleases {EcoRI, Hin&lll and Xbal). The digested DNA was then subjected to electrophoresis on 0.8% agarose gel. DNA fragments were blotted onto a nylon membrane for Southern hybridization, and the membrane was probed with fragments of, and genes. Post-hybridization washes were performed twice succes-

3 60 Ethylene receptors and sex expression in cucumber sively for 5 min each in 2 x SSC/0. % SDS at room temperature and twice successively for 20 min each in 0.5 x SSC/0.% SDS at 65 C. Then the washed membrane was exposed to X-ray film (Hyperfilm-MP, Amersham International pic, Amersham). RNA gel blot analysis Total RNA was extracted from the shoot apices of cucumber as described above. For Northern blotting, 0 fig of total RNA was fractionated by electrophoresis on.0% agarose gel after denaturation with glyoxal at 50 C for h, and the fractionated RNA was then transferred to a nylon membrane. Hybridization was performed as described above. By checking the concentration of rrna stained with ethidium bromide, equal amounts of total RNA were loaded in each lane. Isolation of CS-ACS2 cdna To isolate the CS-ACS2 gene fragment, CS-ACS2 specific primers were synthesized with reference to the nucleotide sequence of CS-ACS2 cdna reported by Kamachi et al. (997). A 730 bp fragment was amplified by RT- PCR using ACC-F, 5-TCGCCGTATTTTGCTGGCTGG-3' and ACC-R, 5-CGCCGAACGTGTACACATCGT-3' as CS-ACS2 specific primers and total RNA isolated from the shoot apices of Higan-fushinari at the 4- stage. The cdnas for RT-PCR were synthesised by Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase RNaseH Minus (Toyobo). The products of PCR were analyzed by gel electrophoresis on 2.0% agarose gel and recovered. The isolated partial cdna product was cloned into the pgem R -T Vector (Promega) by the method of TA cloning. Sequence analysis revealed that the 730 bp fragment was 00% identical to the nucleotide sequence of CS-ACS2 cdna previously reported by Kamachi et al. (997). This CS-ACS2 gene fragment was labeled according to the instructions provided with the DIG RNA Labeling and Detection Kit (Boehringer Mannheim) and used as probe for RNA gel blot analysis. Chemical application To study the effect of ethylene on both sex expression and gene expression, we applied 50 fa of 3.5 x 0 ' mm 2-chloroethylphosphonic acid (Ethrel), an ethylene releasing agent (MacMurray and Miller 968, Iwahori et al. 970), in H 2 O containing 0. % (v/v) Tween 20 to the shoot apices of the monoecious cucumber (Otone No. ) once a day for 3 d. We also applied 50jUl of.0 mm aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene biosynthesis (Adams and Yang 979, Boiler et al. 979), in H 2 O containing 0.% (v/v) Tween 20 to the shoot apices of the gynoecious cucumber (Higan-fushinari) once a day for 3 d. These chemical solutions were applied to shoot apices by using a micropipette when plants were at the 4- stage (i.e., when the blade of the fourth was approximately 2 cm long). Ten gynoecious cucumber plants and twelve monoecious cucumber plants were used for each treatment. The sex of each flower up to node 5 on the main stem was ultimately determined and classified as male or female. Then the percentage of the nodes with male or female flowers on the main stem was calculated. Quantitation of ethylene To examine the time course evolution of ethylene from two cucumber plants, Otone No. and Higan-fushinari, shoot apices were excised at different stages of growth. These samples were each enclosed in a 8.2-ml vessel and sealed with a rubber stopper. After incubation at 25 C for 6 h, ml of head gas was withdrawn from each vessel using a gas-tight syringe and injected into a gas chromatograph (Chromatopac C- R4A, Shimadzu, Kyoto) equipped with a flame ionization detector and an activated alumina column for the measurement of ethylene. The instrument was calibrated with standard ethylene gas. Results Isolation and characterization of cdnas for putative ethylene receptors in cucumber plants To isolate the ethylene-receptor-related genes from cucumber plants, we prepared a set of degenerated oligonucleotides referring to the conserved amino acid sequence found in the ethylene receptor (Chang et al. 993, Wilkinson et al. 995). Three distinct partial cdna fragments of the ethylene receptor were amplified by RT-PCR with the degenerated oligonucleotides and total RNA isolated from the shoot apices of gynoecious cucumber plants, Higan-fushinari, at the 4- stage. The resultant cdna (55 bp) was isolated, subcloned, and sequenced. A homology search revealed that these sequences exhibited strong similarities to those of previously reported ethylene receptors both at the nucleotide and the amino acid level. Full-length cdna clones corresponding to the three partial cdna fragments were isolated from cucumber cdna libraries and named CS- ETR, and. Figure shows a complete amino acid sequence deduced from the nucleotide sequence. The,, and genes encode putative proteins of 740, 767, and 637 amino acids, respectively. Primary sequence analysis indicated that the predicted proteins of,, and exhibit 90%, 7%, and 79% amino acid sequence similarities to Arabidopsis ETR, ETR2, and ERS, respectively. Predicted and proteins consist of an amino-terminal domain, a putative histidine kinase domain, and a putative receiver domain, although the predicted protein has only the amino-terminal domain and the putative histidine kinase domain (Fig. ). Their structural features are thus similar to those of their Arabidopsis homologs. When sequence similarity of the full-length proteins is considered among the cucumber gene products, and are more closely related to each other (85% similarity), and is almost equally distantly related to and (6% and 60% similarity, respectively). The number of genes corresponding to,, and in the two cucumber plants (Otone No. and Higan-fushinari) was estimated by genomic DNA gel blot analysis (Fig. 2). When the or gene was used as a probe, a single band was observed in each lane of the two types of cucumber plants. The band pattern of the fragment was similar to that of the fragment. When a mixed probe of and genes was used, two bands were observed in the EcoRI restriction fragments from the two types of cucumbers. These results indicate that and gene probes hybridized to different DNA fragments. When the gene was used as a probe, one major band accompanied by a minor one was observed

4 Ethylene receptors and sex expression in cucumber 6 -IV- ilkalpsgfl ILLLLASVSA ADNGFPRCNC DDEGSLWSID SIL-ECQRVS -ET CYC IEPQ WPAD ELLMKYQYIS i -EV CNC IEPQ WPAD ELLMKYQYIS IJMES CDC IDA Q WPPD ELLVKYQYIS Ill TYGPHSFQLM LALTVFKILT ALVSCAT@IT LITLIPLLLK VJKVRBFMLKE IFTMHSRTVA WMTTAKVLT AWSCATBLM LVHIIPDLLS VKTRELFLKN TFTTBSRTVA LVMTTAKVLT AWSCATftLM LVHIIPDLLS VRTRELFIiKN TFSMHSKAVA WMTVAKVAC AIVSCATS(LM LVHItPDLLS VKTRBMILKN GLHYCAVWMP NESKTLMNLT HELKDRSFSN GYNVFIPtSD SDVIKIKGSD ALEECALWMP TRTGLELQLS YTLHQQN-PV GYT VPINL PVISQVFSSN ALEECALWMP TRTGLELQLS YTLRHQH-PV EYT VPIQL PVINQVFGTS GU3ECALWMP SRNGLSLQLS HALNYQI-PV GTN IFINL PWNEVFNSN TYTAILVLVL PGGQPRSWNN QBSEIIKWA PQVAVALSHA ALLEESQLMR KRTALMVLMI. PSDSARQWRV HSLELVEWA DQVAVALSH& AILBESMRAR KRXALMVLML PSDSARQWHV HELEXVEWA DQVAVALSHA RSJTAIMVLIL PTDSARKWRD HBLELVDWA DQVAVAL8HA NENMNDD l ILDAMVRTGN WSTQIDDVM EHPIKDSARF PJ.ELEMRSFR ETELTPE L MVETILKSSN LLATLINDVL DLSRLEDG SLQLDIGTFN ETELTPE L MVETILKSSS LLATLMNDVL DLSRLEDG SLQLELGTFN ETELTPE V MIETILKSSN LLATLINDVL DLSRLEDG SLVLDMGSFN fsllndinqg fgyalfrwa ESGSQGRNDQ RVJGNWRQNSS DGDAFIRFEV fnavk-fske fsisisaiva KAETF REI RVPDFHPVPS DSHFYLRVQV ^NAVK-FSKQ SISVTALVT KSDT RAADFFWPT GSHFYLRVKV GNGVK-FTKE HVSIIASVA KLDSL RDW RPTEFYPMQS DGQFYLRVQV NIMVIPNPQG FTRSMALVLR FQLRPSIAVA MPEPGESSEH PHSNSIFR&ti HIWLESEGLG KGCTATFIVK LGIAEQSNES KLPFTSKIHE NSIHTSFPSL Nl»IESDGLG KGCTAIFDVK LGISERSNES KQSGIPKVPA IPRHSNFTSL HIWIESEGPD KGTTAVFIVK LGICNANPND LSVKQWPIV NHRSADLH<3Q *2 LPLHMPELDG FEVTTRIR-K FRSQNY-JtPV IIALTASAGE -DWERCVQIG MDICTPGVDG YELAIRIREK FAK-CHERPF MWLTGNSDK VTKESCLRAS MDVCMPGVEN YQIALRIHEK FTKQRHQftPL LVALSGNTDK STKEKCMSFG X DFLIAVJfijYPS I^IELLYFVS CSNV-PFKWV DFFIALfiifFS IKLELIXFVK KSAVFSYRWV LVQFGJ DFFIAlgjfFS JPiLELItFVK KSAVFPYRWV LVQFG IPILELIXFVQ KSAFFPYRWV LMQJ II_ 23LTHLLHGW CSATHLINLW SATHLINLW 3ATHFINLW KTWDIGREVO MILKQKEAGL HVRHI.TQBIR KS&DRHTILY TTMFELSETL KAAEtDREMG LtRTGEKTGR SVRMLTHE2R ST&DRHtJXK TTLVELGRMj ICAAE DREM<3 LIRTQEBTGR HVRMIiJHEIR STLDRHTILK CTLVELGRTL KAEQLDRKMG LILTQEETGR HVRML2IHEXK STBNRDTLK TILVBXGKTL GVNVLGPNSA LWANCGESD RAVKISPNSP VASLR-PRAG RAVKISPNSP VARLR-PVSG RAICVPYTCQ LARVRTSVGG DKLAEQNRDL QQAKENALMA OPI.MEOWVAI, DLftRREAETA DLLMEetfVAT, DLARREAETA OQLVDQWVAJi DLARREAETA IJJSMIKKAAC LHAVFKBVLN LHTLFREVLN I^IAIFKEALD Gl GIKKSNSQSE K-DTGSGISP K-DSGAGINP K-DSGCGIPP * QVILADADDM KVLVMDDNGV KVLVMOENGV RPIFRETGQV *3 MNGVIRKPVQ MDGLILKPVS LDGVLLKFVS LAITCLCAYKG ilitpvtlvkk I.IKPIAWKK LVKPIASVKK F GSXPNMVS QDIPKLFTKF QDIPKIFTKF QD5HLFTRF NRAVTRKMLE SRSVTKGLI.V SRMVTKGLLV SFSSSRYQRS LQGIAHELRR IDKMRSVLS- LDNIRDVLS- E RGPAAAI RYVAGEWXV KYMLGEWAV RYLPPEWAV SQARUSFQKV NHARUDFLAV IRARNDFLAV IHARNDFLAV FGFAFEVQRS LSLTLHLGLD LPITLNLAPD LSMALILASD GDRRYASD AQT-TVGPRN AQTQSLATRS TQLQTRSNKT RM3PMLRVSNF RVPLLHLSNF RVPLLHLSKF RVPLLNLSNF *_H KSDGMRRPHH MNHEMRTPHH MNHEMRTPMH MNHEMRTPMH LPVFAVODEK LPEFWQ9JJK LPICAVGOEK G2 GAEERIiSFTI SCGSGX.GLAI SGGSGEGLAI NSGVGtGLAL KGGT-PEIVP QINDWEELST QINDWEELST QMNNWPDGSS SIMGLLSMLQ AIIALSSLLQ AIIALSSLfcQ AIIALSSLLL N RVFQVLLHMV RLMQIILNIV RLMQIILNIV CKKLVKLMQO CKRFVNLMEG SKRFVNLHEG KLGCNVTAVS SGFECLTVMA PAGSSIQtfVL HI.(3CEVTTAG 5IEEFI.RVVS QEH KWF HLGCEVTTVS SNEECLRWS HEH KWF L ALLQASKW... ELIERRVLFE TS DliLEPRVLYE GM. Fig. Amino acid sequence alignments of,,, and. Identical amino acids are shaded. Putative transmembrane segments in amino-terminal domains are shown (I, II, III and IV). Amino acids that are mutated in ethylene-insensitive mutants (etrl, Nr) are boxed. The five motifs found in corresponding to those of the bacterial histidine kinases (H, N, Gl, F and G2) are shown. The conserved histidine residue in the H motif, which is postulated to serve as a phosphorylation site in bacteria, is indicated by an asterisk (*). Three residues corresponding to the residues conserved in the bacterial receiver modules are indicated by combinations of numbers (-3) and an asterisk (*) in the EcoRI and Xbal restriction fragments, and a single band was observed in the Hindlll restriction fragments from two cucumber plants. These results suggested that genes corresponding to, and were present as single copies in the genome of both Higanfushinari and Otone No. cucumbers (Fig. 2). A time course study for the expression of, and genes in monoecious and gynoecious cucumber plants We conducted a time course study for the expression of these three ethylene-receptorrelated genes,,, and, in the shoot apices of Higan-fushinari and Otone No. cucumbers to compare their expression with the expression of CS-ACS2 and ethylene evolution. Figure 3 shows the expression of,, and genes in the shoot apices of Higan-fushinari and Otone No. at different stages of growth. There was no significant difference in the accumulation of their mrnas between Higanfushinari and Otone No. at the seedling stage, the 3- stage, or the 8- stage (Fig. 3). On the other hand, accumulation of and transcripts in the shoot apices of the 4- stage and the 5- stage was substantially greater in Higan-fushinari than in Otone No. (Fig. 3). Expression of was also greater in Higan-fushinari than in Otone No., but the difference was not as great as that for the expression of and. A time course study for ethylene evolution and expression of CS-ACS2 gene It has been reported that evolution of ethylene and accumulation of CS-ACS2 mrna in gynoecious cucumber plants were much greater than those in monoecious ones. To test this correlation in the cucumber plants used in the present study, we measured ethylene evolution and the expression of CS-ACS2 in the shoot apices of Higan-fushinari and Otone No. at different stages of growth. Figure 4 shows the time course study for ethylene evolution in the shoot apices of Higanfushinari and Otone No.. The ethylene evolution in the cotyledonary seedlings of both Higan-fushinari and Otone No. was about 0.04^ kg" h" (Fig. 4). The ethylene

5 62 Ethylene receptors and sex expression in cucumber CS-fiTRl (kb) 23. 8= 4.4 (kb) 23. U= 4.4 (kb) 23. a= 4.4 (kb) 23. U= Higan Otone No. -fushinari 3 3 «S Fig. 2 Genomic DNA gel blot analysis with,,, RNA as probes. The DNA from two cucumbers, Otone No. 2.3 and Higan-fushinari, was separately digested with EcoRl, Hindlll, and Xbal. evolution in the shoot apices of Higan-fushinari increased as plants grew from the seedling stage to the 4- stage and decreased from the 4- stage to the 8- stage (Fig. 4). The levels of ethylene evolution at the seedling, 3-, EtBr a Higan -fushinari Otone No. (3D c OJD Hlii^viliii Fig. 3 A time course study for the expression of,, and genes in the shoot apices of two cucumber plants, Higan-fushinari and Otone No.. Total RNA was harvested from the shoot apices of two cucumber plants at the seedling-, 3-, 4-, 5-, and 8- stages. e f - 30 l o * Seedling Higan-fushinari -O- Otone No. 8 () Fig. 4 A time course study of the ethylene evolution from the shoot apices of two cucumber plants, Higan-fushinari and Otone No.. Values indicate the evolution rate of ethylene in the cotyledonary seedlings and shoot apices of plants at the 3-, 4-, 5-, and 8- stages. Vertical bar indicates the standard deviation of the mean for triplicate samples. 4-, 5-, and 8- stages were 0.04, 0.20, 0.26, 0.25, and 0.4//I kg" h", respectively. On the other hand, ethylene evolution in the shoot apices of Otone No. was about 0.5//I kg" h" in the 3- to 5- stages and then decreased to 0.06/dkg"!!" at the 8- stage (Fig. 4). Thus, shoot apices of Higan-fushinari cucumber evolved much more ethylene than those of Otone No. at the 4- and 5- stages (Fig. 4). Figure 5 shows that accumulation of CS-ACS2 mrna in the shoot apices was much greater in Higan-fushinari than in Otone No.. This greater expression of CS-ACS2 was significantly observed at the 4- and 5- stages, cor- C5-AC52 r EtBr Higan -fushinari Otone No. OX) c edli <v iv '' TT IT) *%» II OJD C edli 00 i QO k Fig. 5 A time course study of the expression of CS-ACS2 gene in the shoot apices of two cucumber plants, Higan-fushinari and Otone No.. Total RNA was harvested from the shoot apices of two cucumber plants at the seedling-, 3-, 4-, 5-, and 8- stages.

6 Ethylene receptors and sex expression in cucumber 63 EtBr Otone No. Ethrel Control (3.46x0* mm) w QO HP i.,.<. I ^S ^2 ^B TT 00 TT npip VtflP "** «i* : Wm nm I i 8h (Ethrel/ Control) Fig. 6 Expression of,, and genes as affected by Ethrel application in a monoecious cucumber, Otone No.. The lane "Control" contains RNA from the shoot apices not treated with Ethrel, and the lane "Ethrel" contains RNA from the shoot apices treated with Ethrel (2-chloroethylphosphonic acid at the concentration of 3.46 x 0 ' mm). Ethrel was applied to the shoot apices of cucumber plants at the 4- stage. EtBr Higan-fushinari Control HHHHHI m «lilhhilml^h AVG (l.omm) l I -a ^d QO TT QO Tt mmemm mm WttHm. '. 4d (AVG/ Control) Fig. 7 Expression of,, and genes as affected by AVG application in a gynoecious cucumber, Higanfushinari. The lane "Control" contains RNA from the shoot apices of Higan-fushinari cucumber not treated with AVG. The lane "AVG" contains RNA from the shoot apices of Higanfushinari cucumber treated with AVG at the concentration of.0 mm. AVG was applied to the shoot apices of cucumber plants at the 4- stage. responding to the greater evolution of ethylene. The ethylene-regulated expression of, CS- ETR2, and genes It was shown that expression of ethylene-receptor-related genes, and in particular, correlates with the expression of CS-ACS2 and ethylene evolution. We therefore examined whether ethylene could regulate the expression of, CS- ETR2, and genes in the shoot apices of gynoecious and monoecious cucumber plants. We used Ethrel as an ethylene releasing agent and AVG as an inhibitor of ethylene biosynthesis. Figure 6 shows the effect of Ethrel on gene expression of,, and in the shoot apices of Otone No. at the 4- stage. Compared to the control, the mrna levels of the and genes were significantly elevated 8 h after the start of Ethrel application. When the expression was measured by density, 2.2- and 5.2-fold accumulations of mrna were observed in and, respectively, following Ethrel application. The expression of increased in response to Ethrel application, but it was only.3 fold, much smaller than those of and. (Fig. 6). The increased expression was most remarkable for the gene. The expression of the and CS- ERS genes had decreased to the control level 4 d after the start of Ethrel application (Fig. 6). Figure 7 shows the effect of AVG on gene expression Table Effects of Ethrel and AVG on the sex expression of monoecious (Otone No. ) and gynoecious (Higan-fushinari) cucumber plants Maleflowers (%) Femaleflowers (%) Abortion(%) Otone No. Control Ethrel (3.46x0 'mm) 66.6± ± ± ± ± ±2.2 Higan-fushinari Control AVG (.0 mm) ± ± Each treatment was applied to the shoot apices of cucumber plants at the 4- stage. The percentages of the nodes with male or female flowers were calculated up to the fifteenth node on the main stem. Nodes with no flowers were defined as "abortion".

7 64 Ethylene receptors and sex expression in cucumber of,, and in the shoot apices of Higan-fushinari at the 4- stage. The expression of these genes did not significantly differ from the control 8 h after the commencement of the treatment with AVG (Fig. 7). In contrast, the levels of,, and mrna had all declined 4 d after the treatment with AVG compared to the control (Fig. 7). Effects of Ethrel and A VG application on sex expression Table shows the effects of Ethrel and AVG on the sex expression of cucumber plants. The controls of monoecious cucumber, Otone No., had male tendencies, producing male flowers on 66.6% of the nodes and female flowers on 32.6% of the nodes when observed up to node 5 on the main stem (Table ). When Ethrel was applied to the shoot apices of Otone No. at the 4- stage, female flowers were produced on 72.2% of the nodes, implying a 39.6% increase in femaleness over the control (Table ). The controls of the gynoecious cucumber, Higan-fushinari, produced only female flowers under the present experimental conditions (Table ). However, application of AVG to the shoot apices of Higan-fushinari caused production of male flowers on 34.2% of the nodes (Table ). Discussion It has been suggested that ethylene regulates the sex expression of flowers in cucumber plants (MacMurray and Miller 968, Iwahori et al. 970, George 97, Rudich et al. 972, Beyer 976, Atsmon and Tabbak 979, Takahashi and Suge 980, Takahashi and Jaffe 984, Takahashi and Suge 982, Saito and Takahashi 987). Application of Ethrel, an ethylene releasing agent (MacMurray and Miller 968, Iwahori et al. 970), to the shoot apices of a monoecious cucumber, Otone No., promoted the production of female flowers, whereas a gynoecious cucumber, Higanfushinari, produced male flowers after an application of AVG, an inhibitor of ethylene bioshynthesis (Table ). To investigate the action mechanism of ethylene in the induction of femaleness of cucumber flowers, we isolated the full lengths of three cdna clones,,, and, from cucumber plants, the predicted protein sequences of which were found to be highly homologous to the Arabidopsis putative ethylene receptor, ETR2, and ERS, respectively (Fig. ). While the functions of, and genes have not been established, their significant structural similarity to Arabidopsis suggests that these cucumber homologs likely function as ethylene receptors. When the expression of these ethylene-receptor-related genes,,, and, were analyzed in gynoecious and monoecious cucumbers, it was found that more transcripts accumulated in the gynoecious cucumber than in the monoecious one. Interestingly, the expression pattern of and that of were similar to that of CS-ACS2 gene encoding ACC synthase (Kamachi et al. 997); that is, greater accumulations of and transcripts were observed in the gynoecious cucumber plants at the 4- and 5- stages. These results indicate that the expression of and may be regulated by endogenous ethylene in the shoot apices of cucumber plants. The following results support the hypothesis that ethylene regulates the expression of and. When the effects of Ethrel and AVG on the expression of,, and in the shoot apices of Otone No. and Higanfushinari cucumbers were examined, it was found that Ethrel elevated the mrna levels of and CS- ERS, particularly at 8 h after the start of the treatment (Fig. 6). In contrast, the mrna levels of the and genes in the shoot apices of Higan-fushinari decreased 4 d after the start of the treatment with AVG (Fig. 7). It was reported that mrna levels of the ERS and ETR2 genes were elevated by ethylene application in Arabidopsis (Hua et al. 998). However, role of endogenous ethylene in the accumulation of ERS and ETR2 mrna has not yet been studied. The decreases in the accumulations of and mrna due to AVG application in the present study suggest that ethylene affects the expression of those genes at the endogenous level. The smaller increase in due to ethylene is similar to that of Arabidopsis. Likewise, Nr {Neverripe, LeETR3) mrna is known to be induced by ethylene treatment in mature green fruits of tomato, whereas the expression of Le is not much affected by ethylene (Wilkinson et al. 995, Lashbrook et al. 998). In the present study, accumulation of and mrna, but not of, in the shoot apices of cucumber significantly increased in response to ethylene. In muskmelon, however, the accumulation of Cm- mrna, but not of Cm-ERSl mrna, was more correlated with the climacteric burst of ethylene production during fruit ripening (Sato-Nara et al. 999). Thus, the level of the ERS type of mrna is not always increased by ethylene, and its ethylene regulation may differ among plant species. If this is the case, it is interesting that ethylene influences the expression of ERS type gene differently in cucumber and melon, because both species are member of Cucurbitaceae but their sex expression differs. The former produces male and female flowers in principle, and the latter produces male and hermaphroditic flowers. It has been reported that a strong correlation exists between the evolution of ethylene from the shoot apices and the development of female flowers in cucumber plants (Rudich et al. 972, 976). In addition, the ethylene evolution from the shoot apices of a gynoecious cucumber at the 4- and 5- stages was found to be much greater than that of a monoecious one (Rudich et al. 976). In the present study, this pattern of ethylene evolution was repli-

8 Ethylene receptors and sex expression in cucumber 65 cated in two different cucumber lines, Higan-fushinari and Otone No. (Fig. 4). Since the CS-ACS2 gene was shown to be expressed during the development of female flowers in the apices of gynoecious cucumbers (Kamachi et al. 997), the increase in ethylene evolution by Higanfushinari at the 4- and 5- stages is perhaps attributable to the higher expression of CS-ACS2. Accordingly, we examined the relationship of the expression of CS-ACS2 to ethylene evolution. The results showed that the CS-ACS2 gene was expressed more strongly in the shoot apices of Higan-fushinari than those of Otone No. (Fig. 5). The difference in the expression became obvious at the 4- and 5- stages, at which the shoot apices of Higan-fushinari evolved much more ethylene than that of Otone No. (Fig. 4). Accumulation of CS-ACS2 mrna is probably related to that of and mrna, indicating that not only ethylene biosynthesis but also ethylene reception is involved in the sex expression of flowers in cucumber. Our results therefore suggest that the higher accumulation of and mrna in gynoecious cucumber is attributable to a higher level of endogenous ethylene. It is possible that ethylene-receptor-related genes, and in particular, act at the first step of the ethylene signaling cascade causing the determination of floral sex in cucumber. There is no conclusive evidence for the role of ethylene-receptor-related genes in the differentiation of flower sexes yet, but ethylenereceptor-homolog genes isolated newly in the present study may be useful for the analysis of the action mechanism of ethylene in inducing femaleness in cucumber plants. This work was supported by a Grant-in-aid (094608) from the Ministry of Education, Science, Sports and Culture of Japan to H.T. and the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists to S.Y. This work was also carried out under the Joint Research Program of the Institute of Genetic Ecology, Tohoku University, and the "Ground Research Announcement for Space Utilization" promoted by NAS- DA and the Japan Space Forum. We wish to thank Dr. Atsushi Higashitani of our laboratory for his helpful suggestions and discussion. References Adams, D.O. and Yang, S.F. (979) Ethylene biosynthesis: identification of -aminocyclopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76: Atsmon, D. and Galun, E. (960) A morphogenetic study of staminate, pistillate and hermaphrodite flowers in Cucumis sativus (L.). Phytomorphology 0: 0-5. Atsmon, D. and Tabbak, C. 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