Exogenous Brassinosteroids Influence Sex Expression and Vegetative Development of Cucumber
(Cucumis sativus L.) Plants

Katerina Papadopoulou and Rebecca Grumet

Department of Horticulture, Michigan State University, East Lansing, MI 48824

Additional index words. sex determination, monoecious, flower development

Abstract. Cucumber (Cucumis sativus L.) plants typically exhibit monoecious flowering habit; male flowers are produced first followed by a phase of predominantly female flowers. Hormone balance, especially levels of ethylene and gibberellins, have been shown to influence sexual differentiation of the developing bud. Brassinosterioids are a newly recognized group of endogenous plant hormones that are found in a wide variety of plant species, mainly in flower buds, pollen, fruit, and seeds. Responses to exogenous brassinosteroids include increased cell elongation and division, enhanced epinasty and gravitropism, and altered xylem differentiation. In this study we sought to determine whether brassinosteroids also can influence sex determination of cucumber flowers. Application of 10 or 100 µm epibrassinolide (EBR: 22R,23R,24R-2alpha,3alpha,22,23-tetrahydroxy-B-homo-7-oxa-5alpha-ergostan-6-one) onto the apical meristem of 8-10 day old monoecious cucumber seedlings ('Straight 8'), increased the number of female buds formed per 20 flowering nodes. In addition, increasing concentration of EBR resulted in increasingly earlier appearance of the first female bud. The observed influence of exogenous EBR on total number and time of appearance of the female buds suggests that brassinosteroids may play a role in sex differentiation of cucumber flowers.

We thank Holly Kuehnle and Archana Shenoy for technical assistance. This work was in part supported by USDA­NRI grant no 97-35304-4787 and by the Michigan Agricultural Experimental Station.


Flowering habit in cucumber (Cucumis
L.) is monoecious, with indi-
vidual plants bearing separate male and female flowers. During plant development, male flowers are produced first, followed by a phase of predominantly female flowers (Roy and Saran, 1990). Sex determination of individual flower buds is a response to genetic, environmental and hormonal factors (Roy and Saran, 1990). At early stages after flower initiation male and female buds are morphologically indistinguishable (Goffinet, 1990). Subsequently, the hormonal balance in the vicinity of the bud influences their sexual differentiation.

Several hormones including ethylene, auxins, abscisic acid (ABA) and gibberellins (GAs) have been shown to influence flower sex expression in cucumber. Ethylene appears to be the most important hormonal factor. Application of ethylene or ethylene releasing compounds have been shown

in a variety of studies to increase female flower production in monoecious cucumber, muskmelon (Cucumis melo L.) and squash (Cucurbita pepo L.) cultivars (Augustine et al., 1973; Byers et al., 1972; Rudich et al., 1969, 1972). Consistent with the exogenous effect, the apices and flower buds of gynoecious cucumber and melon plants produced higher ethylene levels compared to monoecious genotypes, suggesting a strong correlation between sex expression and endogenous ethylene production (Byers et al., 1972; Rudich et al., 1972). In contrast, inhibitors of ethylene biosynthesis and action induced staminate flower development in gynoecious lines, and have been used as a tool by breeders to facilitate seed production (Beyers, 1970; Tolla and Peterson, 1979). Recent studies have proposed that ethylene plays a dual role in sex expression, by both promoting femaleness and inhibiting maleness (Yin and Quin, 1995).



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Application of the auxin indole-3-acetic acid (IAA) on cucumber flower buds at the presexual stage, resulted in female flower formation (Galun, 1963). However this effect has been largely attrib uted to increased ethylene production that occurred in response to the IAA application (Takahashi and Jaffe, 1984). Furthermore, the observations that endogenous levels of IAA were found to be lower in gynoecious than monoecious cucumber and squash genotypes (Chrominski and Kopcewicz, 1972; Trebitsh et al., 1987), and that treatment of gynoecious plants with antiauxin compounds did not induce staminate flower formation (Trebitsh et al., 1987), lend additional support to a secondary role of auxins. The role of ABA in floral sex determination, if any, is less clear. Application of ABA to the apices of gynoecious plants enhanced female flower production, but in monoecious lines caused increased male flower formation (Rudich, 1990).

Exogenous gibberellins (GAs) exhibit the opposite effect of ethylene and have been shown to induce formation of male flowers on leaf axils normally occupied by female flowers (Tolla and Peterson, 1979). Higher amounts and activity of endogenous GAs were reported in monoecious compared to gynoecious lines (Atsmon et al., 1968). GAs are also involved in maize sex differentiation, a role which is supported by the GA-deficient mutant phenotype of An1 (anther ear1) and D (Dwarf) genes (Irish, 1996). Mutations in these genes cause derepression of stamen development in ear florets, resulting in hermaphrodite flowers, whereas pistil abortion in tassel florets is unaffected (Lebel-Hardenack et al., 1997). In contrast, mutation in the TASSELSEED2 (TS2) gene leads to the formation of female flowers in the normally male tassel (De Long et al., 1993). GAs have been suggested as potential substrates of the proposed dehydrogenase activity of TS2 (De Long et al., 1993). However, the double mutant phenotype ts2/d1, where hermaphrodite flowers are formed both in tassel and ear, suggests the possibility that TS2 works independently from GAs (Irish, 1996).

Recently, the brassinosteroids (BRs), a new group of polyhydroxysteroid plant hormones, has received increasing attention. BRs are found in a wide variety of species, including dicotyledonous and monocotyledonous, green alga and a fern. They were isolated from plant seeds, fruits, shoots, leaves, flower buds and pollen, at endog

enous levels, sufficient to promote physiological effects (Sakurai and Fujioka, 1993; Takatsuto, 1994; Yokota, 1997).

Although these compounds have been identified and studied for more than a decade, a clear role for endogenous BR has been demonstrated recently, via the identification of Arabidopsis mutants defective in BR biosynthesis and response. The biosynthetic dwarf mutants det2 (deetiolation), cpd (constitutive photomorphogenesis and dwarfism and the response deficient bri1 (brassinosteroid insensitive1) provided strong evidence that BRs play an essential role in several aspects of plant cell division, elongation and photomorphogenesis (Clouse et al., 1996; Kauschmann et al., 1996; Li and Chory, 1997). These mutant genotypes when grown in the light have dark green color, reduced apical dominance and reduced male fertility; while in the dark, these plants exhibited many characteristics of light-grown plants, including cotyledon expansion, primary leaf initiation, anthocyanin accumulation and derepression of light-regulated gene expression. Feeding experiments showed that exogenous BRs, but not GA or auxins, restored the mutant phenotype, (Li et al., 1996; Szekeres et al., 1996).

Biological activity of applied BRs, at nanomolar or micromolar levels, was reported in various bioassay systems designed for GAs, auxins and cytokinins (Takatsuto, 1994). Besides the cell elongation and division in excised stem segments, exogenous BRs were shown to inhibit root growth, promote resistance to stress, enhance gravitropism, induce epinasty, alter xylem differentiation and delay leaf abscission. Further responses to BR application include stimulation of photosynthetic activity, stimulation of membrane hyperpolarization and ATPase activity. Promotion of ethylene biosynthesis via increased ACS (1-aminocyclopropane-1-carboxylic acid synthase) activity also has been reported (Arteca, 1995). In several systems, including cucumber hypocotyl, BRs promoted elongation via synergistic action with auxin (Katsumi, 1985; Kim et al., 1990) and additive action with GAs (Katsumi, 1985).

The pleiotropic effects of BR deficient and response mutants suggest a significant role of BRs on plant development. Based on these multiple

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effects it is reasonable to hypothesize that exogenous BRs might also influence sex differentiation. At this time there is very limited information about this possibility. A role for BRs has been proposed but not tested, in sex determination of corn (Lebel-Hardenack et al., 1997) and an experiment by Suge (1986) indicated that the cucurbit species Luffa cylindrica produced hermaphroditic and pistillate flowers in place of staminate flowers, in response to brassinolide application. In these experiments we seek to examine the potential involvement of BRs in sex differentiation and vegetative development of cucumber plants.

Materials and methods

Seeds of the stable monoecious cucumber genotype 'Straight 8' were pregerminated overnight at 30 oC and planted in 23-cm pots. A preliminary experiment was conducted to test whether BR application on cucumber plants could modify sex differentiation. Cucumber plants were grown in the greenhouse until the first leaf was fully expanded, at which time the synthetic BR, epibrassinolide (22R,23R,24R-2a,3a,22,23-Tetrahydroxy-B-homo-7-oxa-5 a-ergostan-6-on, EBR) (Sigma Chemical Co, St Louis, Mo.) was applied at a concentration of 0.1, 1, and 10 µm, with an atomizer, until runoff.

In the following experiments, to better target the application and reduce the amount of the material used, 250 µL of 0.1, 1, 10, and 100 µm EBR aqueous solution were pipetted onto the apical meristem and the developing leaf. The first application at the first true leaf stage (mean diameter 5 cm), was followed by two subsequent treatments at 3-day

intervals. All the applications were performed in the evening. Observations started 10 days following the last application and were repeated twice at 1-week intervals, until the plants reached the 25th or higher node. The experiments were conducted according to a randomized complete block design (RCB), with 9 replications.

The effect of EBR on sex determination was expressed as the number of female buds formed in 20 flowering nodes and the number of the first female node from the base. Vegetative development was assessed by the plant height at the 25th node and of the 6th, 7th, and 8th internodes.


In the present study, the influence of exogenous BRs on sex differentiation of cucumber flowers was investigated. The 10 and 100 µm levels of EBR caused a significant increase (ANOVA, P < 0.05) in the number of female flowers formed in 20 flowering nodes at 38 days post germination (Fig. 1A, Table 1) as well as a significant decrease (ANOVA, P < 0.05) in the node position of the first female flower bud (Fig. 1B; Table 1, Expts. 1 and 3 and pooled). In addition, the percentage of plants producing female flowers also increased as EBR levels increased (Table 1). Although the lower concentrations of EBR (0.1 and 1µm) did not significantly influence the number of female flowers, but there appeared to be a progressive effect on


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Figure 1. Effect of epibrassinolide (EBR) application on (A) mean number of female buds (data are mean of nine plants/treatment, (SE) at 25, 31, and 38 days postgermination (DPG) and on (B) time to first female flower bud (data presented as the mean number of nodes from the base; each data point is the mean of nine plants (se).


Table 1. The effect of epibrassinolide (EBR) application on the mean number of female flower buds per 20 flowering nodes, on the mean number of node of the first female bud and on the percentage of plants producing female flowers at 4 weeks following treatment. Each value is the mean of nine plants.

Expt. 1z Expt. 2 Expt. 3 Mean (Expts. 1­3)y

First % Plants First % Plants First % Plants First % Plants

EBR No. female with No. female with No. female with No. female with

[µm] females node femalesx females node femalesx females node femalesx females node females

0 0.9 aw 21.9 a 67 0.3 a 24.2 22 0.2 a 27.1 a 33 0.46 24.4 40.6

0.1 1.2 a 19.9 a 67 0.6 a 23.0 33 0.9 a 19.3 ab 44 0.9 20.7 48.0

1 1.4 a 19.0 a 89 0.2 a 22.4 11 1.4 ab 16.6 b 78 1.0 19.3 59.3

10 4.0 b 10.8 b 89 1.5 b 20.2 56 1.6 b 13.5 b 86 2.36 14.8 77.0

100 3.4 b 8.4 b 100 ---v --- --- --- --- --- --- ---

zExperiment illustrated in Figs. 1 and 2.

yData were pooled from Expts. 1­3. Treatment effects were very highly significant (P < 0.05) as determined by analysis of variance of the combined experiments.

xNumber of plants producing female flowers by the 27th, 25th, or 30th node in Expts. 1, 2, and 3, respectively.

wValues followed different letters are significantly different at P = 0.05 by lsd.

vThe 100 µm treatments in Expts. 2 and 3 were eliminated due to plant injury.

time to appearance of the first female flower as EBR concentration increased (r2 = 0.9072, P < 0.005). In Expts. 2 and 3, the 100 µm treatment caused injury to the apex of several plants, so these treatments were eliminated from further analysis. High temperatures in the greenhouse at the time of the EBR application may have contributed to the detrimental effect. Analysis of variance of the combined experiments showed very highly significant effects of the treatment with regard to both the number of female flower buds and the time to appearance of the first female flower bud.

Plant height at the 25th node of the main stem was significantly (ANOVA, P < 0.05) reduced compared to the untreated plants (Fig. 2A). Similarly, length of individual internodes (6, 7, 8) decreased (ANOVA, P < 0.05) as the concentration of the applied EBR increased (Fig. 2B). The leaves within the apical whorl that were directly in contact with the applied EBR showed reduced lamina expansion and petiole elongation and epinasty (Fig. 3). A striking phenotype was apparent increased cell division along the vascular tissue leading to highly undulated veins in the most pronounced cases.



In the present study, application of EBR to cucumber plants revealed a potential role of exogenous BRs in floral sex differentiation. Exogenous BRs induced early and increased production of female buds. The highest concentrations of BRs also resulted in abnormal apical meristem and leaf

development and inhibition of the main stem length.

Earlier work showed that exogenous BRs promote ethylene production via induction of ACS


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Figure 2. Effect of epibrassinolide (EBR) application on (A) the plant height at 25 nodes and on (B) length of the 6th, 7th and 8th internodes.

Figure 3. The effect of epibrassinolide (EBR) application on leaf development and vascular tissue formation. At 100 µm EBR concentration the vascular tissue appeared to have undergone excessive cell division and was highly undulated. This phenomenon was observed in all three experiments.



Although a direct role of BRs in floral sex determination has not been established, it has been suggested that BRs are a potential substrate for the maize sex expression gene TS2 (Lebel-Hardenack et al., 1997). Sequence analysis of TS2, which is essential for female primordia abortion, suggests that it encodes a short-chain alcohol dehydrogenase, with particular similarities to hydroxysteroid dehydrogenases. High expression of TS2 occurs in gynoecium primordia, shortly before the abortion of pistil in male flowers (De Long et al., 1993). Based on BRs hydroxysteroid structure and localization to the stamens (Mandava, 1988), BRs were proposed as potential substrates of TS2 (Lebel-Hardenack et al., 1997). A more direct involvement of BRs in floral development, but not sex expression per se, has been observed in the phenotype of det2 and cpd Arabidopsis mutants, which show reduced male fertility (Clouse et al., 1996). Our results are consistent with and expand upon the observations in Luffa, regarding increased female tendency in response to exogenous BRs (Suge, 1986). In the case of cucumber, however there was full conversion to female flowers rather than production of hermaphrodites.

Future work to study potential role of BRs in sex expression of cucurbits, will include dissection of the exogenous BR effect from ethylene action, study of both female and male fertility of cucumber plants treated with BRs, and the effect on growth, development and sex differentiation of other Cucurbitaceae species.

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