Brassinosteriod-induced Femaleness in Cucumber and Relationship to Ethylene Production

The Cucurbitaceae family is noted for a diversity of sex expression phenotypes. Typically, a phase of male fl owers precedes either female or bisexual fl ower production. Sex determination of individual fl owers is regulated by a combination of genetic, environmental, and hormonal factors. Ethylene, auxins, and gibberellins have all been shown to infl uence fl ower sex expression in cucurbits. Ethylene, which promotes femaleness, plays a predominant role. In this study, we tested whether brassinosteroids (BR), a more recently identifi ed class of plant hormones, also infl uences cucurbit sex expression. Applied epi-brassinolide (epi-BL) caused a signifi cant decrease in time of appearance of the fi rst female fl ower on monoecious cucumber plants, and increased total female fl owers on the main stem. Increasing concentrations had a stronger effect. Of the three species tested, cucumber, melon and zucchini, cucumber was the most responsive to BR. Application of epi-BL also caused an increase in ethylene production by cucumber and zucchini seedlings, suggesting that the BR effect may be mediated by ethylene. To investigate the possible relationship between BR and ethylene on sex expression, we identifi ed the concentration of ethephon (5 ppm) that caused an increase in ethylene production comparable to that induced by 10 μM epi-BL (approximately two-fold). Treatment with 5 ppm ethephon was suffi cient to increase femaleness of cucumber plants, but not zucchini plants, suggesting that the difference in response to epi-BL treatment may refl ect differences in sensitivity to ethylene. Collectively, our results indicate that application of brassinosteroids to cucumber cause earlier and increased female fl ower production, and that the effects may be mediated, at least in part, by brassinosteroid-induced production of ethylene. Flower development is a critical factor infl uencing plant reproduction and crop yield. While most species produce bisexual fl owers possessing both male (stamen) and female (carpel) organs, various species throughout the plant kingdom have evolved mechanisms to produce unisexual fl owers (Ainsworth et al., 1998; Barrett, 1998; Tanurdzic and Banks, 2004). The Cucurbitaceae family is noted for a diversity of sex expression types (Perl-Treves, 1999; Roy and Saran, 1990). Monoecious species, such as cucumber (Cucumis sativus L.) and squash (Cucurbita pepo L.), bear separate male and female fl owers on the same plant, while andromonoecious species, such as many melon genotypes (Cucumis melo L.), produce male and bisexual fl owers. In both cases, male fl owers are produced early in plant development, followed by a mixture of either female and male, or bisexual and male fl owers. Sex determination of individual fl ower buds is regulated by a combination of genetic, environmental, and hormonal factors. At the initial stages of fl oral primordium development, male and female buds are morphologically indistinguishable; all four whorls (sepals, petals, stamens, and carpels) are initiated, and subsequently, either male or female organs develop (Goffi net, 1990). Studies with homeotic mutants of cucumber indicate that position thase), a key enzyme in ethylene biosynthesis, showed increased femaleness (Papadopoulou et al., 2005). Conversely, inhibitors of ethylene biosynthesis and action promote staminate fl ower development, and have been used by breeders to facilitate seed production on gynoecious genotypes (Byers et al., 1972; Den Nijs and Visser, 1980; Owens et al., 1980; Takahashi and Jaffe, 1984; Tolla and Peterson, 1979). Transgenic melon constitutively expressing the Arabidopsis mutant ethylene perception gene, etr1-1, failed to produce female fl owers (Papadopoulou et al., 2002). Application of auxin to cucumber fl ower buds at the pre-sexual stage also can increase female fl ower formation (Galun et al., 1963; Trebitsh et al., 1987). However, fi ndings that endogenous IAA levels were lower in gynoecious than monoecious cucumber and squash genotypes (Chrominski and Kapcewicz, 1972; Trebitsh et al., 1987), and that treatment of gynoecious plant with anti-auxin compounds did not induce staminate fl ower production (Trebitsh et al., 1987), suggested a secondary role for auxins. Instead, it has been proposed that the effect of auxin is mediated by increased ethylene production that occurs in response to auxin application (Takahashi and Jaffe, 1984; Trebitsh et al., 1987). Exogenous GAs exhibit the opposite effect of ethylene, and have been shown to induce formation of male fl owers on leaf axils normally occupied by female fl owers (Pike and Peterson, 1969; Tolla and Peterson, 1979). Higher levels and activity of GAs were reported in monoecious compared to gynoecious lines (Atsmon et al., 1968; Freidlander et al., 1977). However, GA effects also appear to be secondary to ethylene. Studies by Yin and Quinn (1995), comparing the effects of ethylene and GA concluded that ethylene is the predominant sex determining hormone, and Tolla and Peterson (1979) observed that inhibition of ethylene action was more effective in inducing male fl ower production in cucumber than was application of gibberellins. In the past decade, a new group of plant hormones, the brassinosteroids, which can promote cell division and expansion, vascular differentiation, reproductive development, senescence, photomorphogenesis, and plant tolerance to stress, has received increasing attention (Clouse and Sasse, 1998; Sasse, 1997). A defi nitive role for endogenous brassinosteroids in plant growth and development was demonstrated by the identifi cation of developmental mutants of Arabidopsis that are defective in brassinosteroid biosynthesis and response (Altmann, 1999; Clouse et al., 1996; Clouse, 1997; Kauschmann et al., 1996; Li and Chory, 1999). Given the range of activities of brassinosteroids, and the role of several hormones in infl uencing sex determination in cucurbits, we were interested in testing whether exogenous brassinosteroid would affect sex expression in cucurbits. Our results demonstrate that application of brassinosteroid to cucumber caused earlier and increased female fl ower production and increased ethylene production, and suggest that the effects of brassinosteroid within the developing fl ower bud (i.e., third or fourth whorl) is critical for determining whether the specifi c fl oral organ will develop (Kater et al., 2001). Several plant hormones, including ethylene, auxins, and gibberellins (GAs) have been shown to infl uence fl ower sex expression in cucurbits (Rudich, 1990). Application of exogenous hormones at the appropriate time can cause fl oral primordia destined to become unisexual fl owers to be converted to bisexual fl owers, or completely change to the opposite sex. The extent of conversion can be affected by genotype, stage of fl oral development at the time of application, and amount of applied hormone. Application of ethylene or ethylene releasing compounds have been shown to increase pistillate fl ower production in cucumber, squash and muskmelon (Augustine et al., 1973; Hume and Lovell, 1983; Karchi, 1970; McMurray and Miller, 1968, Owens, 1980; Robinson et al., 1969; Rudich et al., 1969, 1972). Consistent with exogenous effects, excised apices and fl ower buds of gynoecious (all female) cucumber plants produced 2to 3fold higher ethylene levels compared to monoecious genotypes, suggesting a correlation between sex expression and endogenous ethylene production (Byers et al., 1972a; Rudich et al., 1972, 1976; Trebitsh et al., 1987; Yamasaki et al., 2001). Although such a difference was not observed in melon, hypobaric conditions reducing ethylene levels increased maleness (Byers et al., 1972b) and transgenic melon plants constitutively expressing ACS (1-amino-cyclopropane-1-carboxylic acid synHORTSCIENCE 40(6):1763–1767. 2005. Received for publication 5 May 2005. Accepted for publication 3 June 2005. We thank Randy Beaudry and David Dilley for providing use of gas chromatography equipment and advice for the ethylene measurements and Amy Iezzoni and Randy Beaudry for helpful reviews of the manuscript. This project was in part supported by BARD grant IS-3139-99. 937-Growth.indd 1763 8/18/05 9:00:57 AM HORTSCIENCE VOL. 40(6) OCTOBER 2005 1764 may be mediated, at least in part, via increased production of ethylene. Materials and Methods Plant materials. The genotypes used were monoecious cucumber cultivar Straight 8 (S8) (Hollar Seeds, Rocky Ford, Colo.), the gynoecious cucumber breeding line GY14 (kindly provided by Greg Tolla, Seminis Seeds), the monoecious zucchini cultivar Black Beauty (Willhite, Poolville, Texas), and the andromonoecious melon cultivar Hale’s Best Jumbo (Hollar Seeds). For greenhouse trials, seeds were imbibed overnight at 30 °C before planting in 23-cm-diameter pots containing Baccto soil mix (70% to 80% sphagnum peat, pH 5.5 to 6.5; Baccto, Michigan Peat Co., Houston, Texas). Plants were grown in the greenhouse throughout the year under natural photoperiods at average temperatures of 25 °C, and fertilized once a week with 300 ppm 20–20–20 (Peter’s Special). Brassinosteroid experiments. The synthetic brassinosteroid, epibrassinolide (epi-BL; 22R, 23R, 24R2α, 3α, 22,23-tetrahydroxy-B-homo7-oxa-5α-ergostan-6-one) (Sigma Chemical Co., St Louis, Mo.) was applied by pipetting 250 μL of 0.1, 1, or 10 μM epiBL onto the apical meristem and developing leaf. The fi rst application was made at the fi rst true leaf stage (mean diameter about 5 cm); two subsequent treatments were applied at three day intervals. Plants were scored for presence of pistillate buds on the main stem. The experiments were arranged in a randomized complete block design with 5 to 10 replications per treatment, as specifi ed in fi gure legends. Each experiment was repeated three or four times. Ethylene experiments. Cucumber (Cucumis sativus L. ‘Straight 8’) or zucchini (C. pepo ‘Black Beauty’) seeds were de-coated, sterilized in LD solution (Alcide LD, Alcide Corporation, Norwalk, Conn.) for 10 min, rinsed with distilled water three times, and transferred to 250-mL plastic containers (Magenta boxes, Magenta Corp., Chicago Ill.) containing medium with Murashige and Skoog basal salts (Sigma), 3% sucrose, 0.8% agar, adjusted to pH 5.7 to 5.8, and placed under 16 h light at 25 °C. Each box contained six seedlings. At 8 to 10 d old (cotyledons fully expanded, fi rst true leaf just emerging), seedlings were treated with 20 μL of 0, 1, and 10 μM epi-BL. Headspace gas samples were taken with an airtight syringe at 0, 6, 12, 24, 48, and 72 h postapplication. The samples were analyzed in a gas chromatographer (HACH CARLE series 100 AGC, Linear 1200 recorder, with an alumina column and fl ame ionization detector). The same experimental procedure was used to determine the concentration of ethephon (2-chloroethyl phosphonic acid) that releases the same amount of ethylene as was caused by application of 10 μM epi-BL. Ethephon was applied at concentrations of 5, 50, and 500 ppm. Numbers of replications per experiment are indicated in the fi gure legend. Each experiment was repeated three times. The greenhouse experiments were performed as described above for epi-BL, using 5 ppm ethephon; each experiment was repeated four times.