Characterization of ethylene effects on sex determination in cucumber plants

Sex differentiation in cucumber plants (Cucumis sativus L.) appears to be determined by the selective arrest of the stamen or pistil primordia. We investigated the influence of an ethylene-releasing agent (ethephon) or an inhibitor of ethylene biosynthesis (aminoethoxyvinyl glycine) on sex differentiation in different developmental stages of flower buds. These treatments influence sex determination only at the stamen primordia differentiation stage in both monoecious and gynoecious cucumbers. To clarify the relationships between the ethylene-producing tissues and the ethylene-perceiving tissues in inducing female flowers in the cucumber, we examined the localization of mRNA accumulation of both the ACC synthase gene (CS-ACS2) and the ethylene-receptor-related genes (CS-ETR1, CS-ETR2, and CS-ERS) in flower buds by in situ hybridization analysis. CS-ACS2 mRNA was detected in the pistil primordia of gynoecious cucumbers, whereas it was located in the tissues just below the pistil primordia and at the adaxial side of the petals in monoecious cucumbers. In flower buds of andromonoecious cucumbers, only CS-ETR1 mRNA was detected, and was located in the pistil primordia. The localization of the mRNAs of the three ethylene-receptor-related genes in the flower buds of monoecious and gynoecious cucumbers overlap but are not identical. We discuss the relationship between the mRNA accumulation patterns and sex expression in cucumber plants.     

Adventitious staminate flower formation in gibberellin treated gynoecious cucumber plants

Single gibberellin (A₄₊₇) treatments induced the appearanceof staminate floral buds in several consecutive nodes on themain stem of genetically female cucumber (Cucumis sativus L.).The staminate buds appeared next to pistillate buds which showedvarious degrees of degeneration. Similarly, repeated GA treatmentsinduced the appearance of staminate flowers in otherwise strictlyhermaphrodite plants, next to bisexual flowers. However, thebisexual buds, unlike the pistillate ones, did not show anydeleterious effects of the GA treatment. Therefore, it is inferredthat the hormonally induced staminate buds did not develop bysexual reversion of would-be pistillate or bisexual buds, butrather, represent adventitious buds which, in normally grownfemale or hermaphrodite plants, never develop. It thus seemsthat predetermined pistillate or bisexual buds do not changeinto staminate ones, while change in the reverse direction hasbeen demonstrated in the past (at least for the gynoecious ones). The effectiveness of the GA treatment in the gynoecious plantsshowed an acropetal gradient both within the affected region,as well as along the main stem. Autoradiographic histologicalexaminations showed that the course of development of the inducedstaminate floral bud did not differ from that of normally developingbuds. (Received June 16, 1977; )     

The M locus and ethylene-controlled sex determination in andromonoecious cucumber plants.

Sex determination in cucumber (Cucumis sativus L.) plants is genetically controlled by the F and M loci. These loci interact to produce three different sexual phenotypes: gynoecious (M-F-), monoecious (M-ff), and andromonoecious (mmff). Gynoecious cucumber plants produce more ethylene than do monoecious plants. We found that the levels of ethylene production and the accumulation of CS-ACS2 mRNA in andromonoecious cucumber plants did not differ from those in monoecious plants and were lower than the levels measured in gynoecious plants. Ethylene inhibited stamen development in gynoecious cucumbers but not in andromonoecious ones. Furthermore, ethylene caused substantial increases in the accumulation of CS-ETR2, CS-ERS, and CS-ACS2 mRNA in monoecious and gynoecious cucumber plants, but not in andromonoecious one. In addition, the inhibitory effect of ethylene on hypocotyl elongation in andromonoecious cucumber plants was less than that in monoecious and gynoecious plants. These results suggest that ethylene responses in andromonoecious cucumber plants are reduced from those in monoecious and gynoecious plants. This is the first evidence that ethylene signals may influence the product of the M locus and thus inhibit stamen development in cucumber. The andromonoecious line provides novel material for studying the function of the M locus during sex determination in flowering cucumbers.     

Ethylene evolution from cucumber plants as related to sex expression

Ethylene evolved from monoecious and gynoecious cucumber (Cucumis sativus) plants grown under short and long day conditions was determined. More ethylene was evolved from floral buds and apices bearing buds than from whole seedlings of comparable weight. More ethylene also was evolved from apices of the gynoecious than from those of the monoecious type. Furthermore, quantities evolved from female buds were greater than from male ones and plants grown under short day conditions which promote femaleness evolved more ethylene than those grown under long day conditions. The data suggest that ethylene participates in the endogenous regulation of sex expression by promoting femaleness.     

Stamen development in <Emphasis Type="Italic">Arabidopsis</Emphasis> is arrested by organ-specific overexpression of a cucumber ethylene synthesis gene <Emphasis Type="Italic">CsACO2</Emphasis>

Cucumber (Cucumis sativus L.) has served as a model to understand hormone regulation in unisexual flower development since the 1950s and the role of ethylene in promoting female flower development has been well documented. Recent studies cloned the F-locus in gynoecious lines as an additional copy of the ACC synthase (ACS) gene, which further confirmed the role of ethylene in the promotion of female cucumber flowers. However, no direct evidence was generated to demonstrate that increases in endogenous ethylene production could induce female flowers by arresting stamen development. To clarify the relationship between ethylene production and stamen development, we overexpressed the ethylene synthesis cucumber gene CsACO2 to generate transgenic Arabidopsis, driven by the organ-specific promoter P _AP3 . We found that organ-specific overexpression of CsACO2 significantly affected stamen but not carpel development, similar to that in the female flowers of cucumber. Our results suggested that increases in ethylene production directly disturb stamen development. Additionally, our study revealed that among all floral organs, stamens respond most sensitively to exogenous ethylene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Floral primordia-targeted ACS (1-aminocyclopropane-1-carboxylate synthase) expression in transgenic Cucumis melo implicates fine tuning of ethylene production mediating unisexual flower development

Main conclusion

Floral primordia-targeted expression of the ethylene biosynthetic gene, ACS , in melon suggests that differential timing and ethylene response thresholds combine to promote carpels, inhibit stamens, and prevent asexual bud formation. Typical angiosperm flowers produce both male and female reproductive organs. However, numerous species have evolved unisexuality. Melons (Cucumis melo L.) can produce varying combinations of male, female or bisexual flowers. Regardless of final sex, floral development begins with sequential initiation of all four floral whorls; unisexuality results from carpel or stamen primordia arrest regulated by the G and A loci, respectively. Ethylene, which promotes femaleness, is a key factor regulating sex expression. We sought to further understand the location, timing, level, and relationship to sex gene expression required for ethylene to promote carpel development or inhibit stamen development. Andromonoecious melons (GGaa) were transformed with the ethylene biosynthetic enzyme gene, ACS (1-aminocyclopropane-1-carboxylate synthase), targeted for expression in stamen and petal, or carpel and nectary, primordia using Arabidopsis APETALA3 (AP3) or CRABSCLAW (CRC) promoters, respectively. CRC::ACS plants did not exhibit altered sex phenotype. AP3::ACS melons showed increased femaleness manifested by gain of a bisexual-only phase not seen in wild type, decreased male buds and flowers, and loss of the initial male-only phase. In extreme cases, plants became phenotypically hermaphrodite, rather than andromonoecious. A reduced portion of buds progressed beyond initial whorl formation. Both the ACS transgene and exogenous ethylene reduced the expression of the native carpel-suppressing gene, G, while elevating expression of the stamen-suppressing gene, A. These results show ethylene-mediated regulation of key sex expression genes and suggest a mechanism by which temporally regulated ethylene production and differential ethylene response thresholds can promote carpels, inhibit stamens, and prevent the formation of asexual buds.     

Epigenetic control of sexual phenotype in a dioecious plant,Melandrium album

Melandrium album (syn.Silene latifolia) is a model dioecious species in which theY chromosome, present only in heterogametic males, plays both a male-determining and a strict female-suppressing role. We showed that treatment with 5-azacytidine (5-azaC) induces a sex change to androhermaphroditism (andromonoecy) in about 21% of male plants, while no apparent phenotypic effect was observed in females. All of these bisexual androhermaphrodites (with the standard male 24,AA +XY karyotype) were mosaics possessing both male and hermaphrodite flowers and, moreover, the hermaphrodite flowers displayed various degrees of gynoecium development and seed setting. Southern hybridization analysis with a repetitive DNA probe showed that the 5-azacytidine-treated plants were significantly hypomethylated in CG doublets, but only to a minor degree in CNG triplets. The bisexual trait was transmitted to two successive generations, but only when androhermaphrodite plants were used as pollen donors. The sex reversal was inherited with incomplete penetrance and varying expressivity. Based on the uniparental inheritance pattern of androhermaphroditism we conclude that it originated either by 5-azaC induced inhibition ofY-linked female-suppressing genes or by a heritable activation of autosomal female-determining/promoting genes which can be reversed, on passage through female meiosis, by a genomic imprinting mechanism. The data presented indicate that female sex suppression inM. album XY males is dependent on methylation of specific DNA sequences and can be heritably modified by hypomethylating drugs.     

Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants

Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is associated with the differentiation and development of female flowers.     

罗汉果花芽分化过程中形态及其激素水平变化特征

采用石蜡切片和酶联免疫法(ELISA)对罗汉果雄性、雌性、两性花芽分化过程的形态和激素水平变化进行观测,为罗汉果开花调控和品种选育提供科学依据。结果表明:(1)罗汉果雄性、雌性、两性花的花芽分化过程均可分为花芽未分化期、花芽分化初期、花序分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基分化期和雌蕊原基分化期7个阶段。雄蕊原基分化期前,3种花芽分化过程无明显差异,各时期形态特征均依次为:茎端呈圆锥状(花芽未分化期)→茎端经半球形变成扁平状(花芽分化初期)→距茎端5~7节位处分化出穗状花序(花序分化期)→小花原基周围形成5个萼片原基(萼片原基分化期)→萼片原基内侧形成5个花瓣原基(花瓣原基分化期)。雄蕊和雌蕊原基分化期,3种花芽分化过程存在明显差异,雄蕊原基内侧出现雌蕊原基后,雄花芽雄蕊原基继续发育成雄蕊,雌蕊原基停滞生长,退为一个小突起;雌花芽雌蕊原基继续发育成雌蕊,雄蕊原基生长缓慢,退化为小花丝;两性花芽雌蕊和雄蕊原基均继续发育,形成外观正常的雌蕊和雄蕊。(2)内源激素脱落酸(ABA)、赤霉素(GAs)和玉米素核苷(ZR)含量在3种花芽分化过程中变化规律相似,即ABA含量在花芽生理分化期降低,花芽形态分化期升高,而GAs和ZR含量则基本保持不变;吲哚乙酸(IAA)含量在3种花芽分化过程中变化存在明显差异,雌花芽IAA含量在花芽生理分化期升高,花芽形态分化期逐渐降低,而雄性和两性花芽的IAA含量则基本保持不变。ABA/GAs、ABA/IAA、ZR/IAA和ZR/GAs激素含量比值在3种花芽分化过程中变化规律相似,ABA/GAs在花芽生理分化期降低,花芽形态分化期升高,而BA/IAA、ZR/IAA和ZR/GAs则基本保持不变。研究认为,罗汉果花芽分化过程经历一个"两性期",高ABA含量和ABA/GAs比值有利于罗汉果花芽分化,IAA可能对罗汉果花性分化具有重要作用。     

The influence of ethylene perception on sex expression in melon (<Emphasis Type="Italic">Cucumis melo </Emphasis>L.) as assessed by expression of the mutant ethylene receptor, <Emphasis Type="Italic">At-etr1-1</Emphasis>, under the control of constitutive and floral targeted promoters

Sexual diversity expressed by Curcurbitaceae species is a primary example of developmental plasticity in plants. Ethylene, which promotes femaleness (carpel development), plays a key role in sex determination. We sought to determine the critical location for ethylene perception in developing floral primodia. The dominant negative Arabidopsis ethylene response mutant gene, etr1-1, was introduced into melon (Cucumis melo L.) plants under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, or floral-targeted Apetela3 (AP3) and Crab’s Claw (CRC) promoters, which in Arabidopsis, promote expression in petal and stamen, and carpel and nectary primordia, respectively. Based on effects of exogenous ethylene, it was predicted that inhibition of ethylene perception by carpel primordia would inhibit carpel development. Constitutive expression of etr1-1 caused several phenotypes associated with ethylene insensitivity, verifying that etr1-1 inhibits ethylene perception in the heterologous melon system. Carpel-bearing bud production was essentially abolished in 35S::etr1-1 melons, providing direct demonstration of the requirement for ethylene perception for carpel development. CRC::etr1-1 plants, however, showed enhanced femaleness as manifested by earlier and increased number of carpel-bearing buds, and production of female (rather than bisexual) buds. Despite increased carpel-bearing bud formation, a greater proportion of the CRC::etr1-1 carpel-bearing buds aborted before anthesis. AP3::etr1-1 plants showed increased maleness by nearly exclusive staminate flower production, and poorly developed carpels in the rare bisexual flowers. These results indicate that ethylene perception by the stamen (or petal) primordia plays a critical role in promoting carpel development at the time of sex determination, while ethylene perception by the carpel is important for maturation of carpel-bearing flowers to anthesis.     

Sex determination in the monoecious species cucumber is confined to specific floral whorls

In unisexual flowers, sex is determined by the selective repression of growth or the abortion of either male or female reproductive organs. The mechanism by which this process is controlled in plants is still poorly understood. Because it is known that the identity of reproductive organs in plants is controlled by homeotic genes belonging to the MADS box gene family, we analyzed floral homeotic mutants from cucumber, a species that bears both male and female flowers on the same individual. To study the characteristics of sex determination in more detail, we produced mutants similar to class A and C homeotic mutants from well-characterized hermaphrodite species such as Arabidopsis by ectopically expressing and suppressing the cucumber gene CUCUMBER MADS1 (CUM1). The cucumber mutant green petals (gp) corresponds to the previously characterized B mutants from several species and appeared to be caused by a deletion of 15 amino acid residues in the coding region of the class B MADS box gene CUM26. These homeotic mutants reveal two important concepts that govern sex determination in cucumber. First, the arrest of either male or female organ development is dependent on their positions in the flower and is not associated with their sexual identity. Second, the data presented here strongly suggest that the class C homeotic function is required for the position-dependent arrest of reproductive organs.     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.