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.     

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.     

Identification of a 1-aminocyclopropane-1-carboxylic acid synthase gene linked to the female (F) locus that enhances female sex expression in cucumber.

Sex determination in cucumber (Cucumis sativus L.) is controlled largely by three genes: F, m, and a. The F and m loci interact to produce monoecious (M_f_) or gynoecious (M_f_) sex phenotypes. Ethylene and factors that induce ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylate (ACC) and auxin, also enhance female sex expression. A genomic sequence (CS-ACS1) encoding ACC synthase was amplified from genomic DNA by a polymerase chain reaction using degenerate oligonucleotide primers. Expression of CS-ACS1 is induced by auxin, but not by ACC, in wounded and intact shoot apices. Southern blo hybridization analysis of near-isogenic gynoecious (MMFF) and monoecious (MMff) lines derived from divers genetic backgrounds revealed the existence of an additional ACC synthase (CS-ACS1G) genomic sequence in the gynoecious lines. Sex phenotype analysis of a segregating F2 population detected a 100% correlation between the CS-ACS1G marker and the presence of the F locus. The CS-ACS1G gene is located in linkage group B coincident with the F locus, and in the population tested there was no recombination between the CS-ACS1G gene and the F locus. Collectively, these data suggest that CS-ACS1G is closely linked to the F locus and may play a pivotal role in the determination of sex in cucumber flowers.     

Isolation of a partial sequence of a putative nucleotide sugar epimerase, which may involve in stamen development in cucumber (Cucumis sativus L.)

Sex determination is the most widely studied subject in cucumber. The sex of cucumber plants can be monoecious, hermaphrodite, gynoecious, androecious, or andromonoecious. Besides environmental factors, three major genes, F/f, M/m, and A/a mainly govern the sex types in cucumber. Regardless of their sex all floral buds are bisexual at the early bud stage. A stage specific arrest of either stamen or carpel leads to unisexual flower development. The possible downstream product of the interaction of the sex determining genes that may directly allow the growth or selectively arrest stamen or pistil is not yet identified. Therefore, in the current study, we performed suppression subtractive hybridization using floral buds from nearly isogenic gynoecious and hermaphrodite cucumber plants and identified for the first time a cDNA homologous to nucleotide sugar epimerase. The expression level of the isolated putative nucleotide sugar epimerase is weak in female floral buds but strong in bisexual and male flowers. The weak level of the putative nucleotide sugar epimerase may be an indication for its improper functioning, which may influence stamen development in cucumber plants.     

Photoperiodic regulation of CS-ACS2, CS-ACS4 and CS-ERS gene expression contributes to the femaleness of cucumber flowers through diurnal ethylene production under short-day conditions

Photoperiod and the plant hormone, ethylene, modify sex expression of flowers in cucumber (Cucumis sativus L.). In the present study, femaleness of cucumber occurred under short‐day (8 h photoperiod) conditions compared to that under long‐day (16 h photoperiod) conditions, although the effect of photoperiod was more pronounced in a monoecious than in an andromonoecious cucumber. Application of ethylene had a greater effect than photoperiod on the production of female and bisexual flowers in monoecious and andromonoecious cucumbers, respectively. Ethylene evolution and the expression of CS‐ACS2, CS‐ACS4 and CS‐ERS genes in the shoot apices of both monoecious and andromonoecious cucumber plants had a diurnal rhythm with a peak in the middle of an 8 h or a 16 h light period. Peak ethylene evolution and expression of CS‐ACS2 was greater under short‐day conditions than under long‐day conditions in a monoecious cucumber but not in an andromonoecious one. Expression of CS‐ACS4 in monoecious and andromonoecious cucumber plants did not differ, but the level was higher under short‐day conditions compared with that under long‐day conditions. Thus, CS‐ACS2 and CS‐ACS4 might be involved in the basic diurnal rhythm of ethylene evolution in cucumber. Because exogenous ethylene increased the expression of CS‐ACS2 and CS‐ERS in monoecious cucumber possessing the M locus, but not in andromonoecious cucumber in which the function of the M locus was lost (Yamasaki et al. Plant and Cell Physiology 42, 608–619, 2001), the CS‐ACS2 gene might also be involved in ethylene production by positive feedback via regulation of M locus under short‐day conditions.     

The level of phytohormones in monoecious and gynoecious cucumbers as affected by photoperiod and ethephon

The endogenous levels of auxin, gibberellin, and inhibitors were followed in monoecious and gynoecious cucumber (Cucumis sativus L.) plants, and in plants treated with the ethylene-releasing compound Ethephon (2-chloroethyl phosphonic acid). Higher auxin inhibitor and lower gibberellin levels were associated with female tendency. The endogenous level of gibberellin and auxin decreased in Ethephon-treated plants. Application of Ethephon induced a rise in abscisic acid. Root application of abscisic acid promoted female tendency of gynoecious cucumbers grown under conditions which increase maleness. High CO₂ levels, which are known to antagonize ethylene, increased maleness of gynoecious cucumbers. The possibility of interrelationship between gibberellin, auxin, ethylene, and abscisic acid on sex expression are discussed.     

Sexual differentiation in cucumber: The effects of abscisic acid and other growth regulators on various sex genotypes

AbA, ethephon and gibberellin were applied to cucumber plantsof monoecious, gynoecious, andromoneocious and hermaphroditeinbred lines, as well as to F₁ (gynoecious?monoecious) plants.Exogenous AbA enhanced the male tendency in monoecious cucumberplants and the female tendency in gynoecious plants, irrespectiveof light regime. Exogenous ethephon treatments increased thefemale tendency in monoecious plants, and decreased it in gynoeciousones. These effects were influenced by day length. ExogenousAbA counteracted the effect of gibberellin (A₄₊₇) treatmentin gynoecious plants, but had no such effect in monoecious ones. In addition to its differential effect on sexual differentiation,AbA stimulated flower development in gynoecious plants and inhibitedit in monoecious plants. These responses to AbA are discussedin the light of previously reported effects of plant growthregulators on various sex types of cucumber. The present resultsare being integrated into an updated working hypothesis on sexcontrol in cucumbers. (Received August 30, 1976; )     

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.     

Auxin,biosynthesis of ethylene and sex expression in cucumber (Cucumis sativus)

Ethylene production, level of 1-aminocyclopropane-1-carboxylic acid (ACC) and activity of the ethylene forming enzyme (EFE) were higher in apices of gynoecious cucumber (Cucumis sativus cv. Alma) as compared to monoecious cucumber (C. sativus cv. Elem). Application of indole-3-acetic acid (IAA) enhanced ethylene and ACC production in both cultivars. The stimulatory effect of IAA was more pronounced in gynoecious apices. Induction of ethylene production and accumulation of ACC resulting from treatment with IAA were effectively blocked by aminoethoxyvinylglycine (AVG). Content of endogenous IAA, measured by an enzyme immunoassay, was lower in gynoecious cucumber as compared to monoecious one. Treatment of gynoecious plants with the antiauxins -(p-chlorophenoxy)isobutyric acid (PCIB) and -naphthaleneacetic acid (-NAA) did not inhibit female sex expression.It appears that although exogenous IAA enhances ACC and ethylene production, endogenous IAA might not have a major role in the control of sex expression in cucumber of the Beit-Alfa type.Prof. Rudich passed away in May 1986.     

Peroxidase and IAA Oxidase Activity and Isoenzyme Patterns in Cucumber Plants, as Affected by Sex Expression and Ethephon

Peroxidase and IAA-oxidase activity, electrophoretic patterns of total proteins and isoenzymes and the effect of Ethephon (= Ethrel, 2-chloroethane phosphonic acid) on these patterns were compared in extracts of monoecious and gynoecious cucumber plants. The activity of both peroxidase and IAA oxidase was greater in gynoecious than in monoecious plants. Ethephon treatments given at the 2-leaf stage increased peroxidase activity, especially in monoecious plants, and decreased IAA-oxidase activity. Ethephon treatment did not affect total protein or isoenzyme patterns, but increased band intensity, especially that of one band in monoecious plant extracts. No differences between monoecious and gynoecious plants were found in total protein, peroxidase and IAA-oxidase isoenzyme patterns. Peroxidase and IAA-oxidase activity sites on the gel were similar.     

Some Morphogenic Differences between Monoecious and Gynoecious Cucumber Seedlings as Related to Ethylene Production

Exposure of gibberellic acid-treated seedlings of a monoecious cucumber cultivar `Chipper' (Cucumis sativus L.) to ethylene caused thickening of the hypocotyl, inhibited longitudinal growth, and had no effect on fresh weight. Downward curvature of cotyledons was increased by the presence of ethylene. A gynoecious breeding line, `Gy 3,' had thicker hypocotyls and displayed its cotyledons in a more downward position than `Chipper'. Excised hypocotyls of the gynoecious seedlings produced three times as much ethylene as did the monoecious Chipper hypocotyls. Thus, ethylene may play a role in the regulation of cucumber seedling morphology.     

Mapping of the Gynoecy in Bitter Gourd (Momordica charantia) Using RAD-Seq Analysis

Momordica charantia is a monoecious plant of the Cucurbitaceae family that has both male and female unisexual flowers. Its unique gynoecious line, OHB61-5, is essential as a maternal parent in the production of F₁ cultivars. To identify the DNA markers for this gynoecy, a RAD-seq (restriction-associated DNA tag sequencing) analysis was employed to reveal genome-wide DNA polymorphisms and to genotype the F₂ progeny from a cross between OHB61-5 and a monoecious line. Based on a RAD-seq analysis of F₂ individuals, a linkage map was constructed using 552 co-dominant markers. In addition, after analyzing the pooled genomic DNA from monoecious or gynoecious F₂ plants, several SNP loci that are genetically linked to gynoecy were identified. GTFL-1, the closest SNP locus to the putative gynoecious locus, was converted to a conventional DNA marker using invader assay technology, which is applicable to the marker-assisted selection of gynoecy in M. charantia breeding.     

黄瓜茎端ABA/GA_4比值与花芽性别分化的关系(英文)

在同一环境条件下,单雌性株茎端ABA含量显著高于雌雄同株的,而GA_4水平却正好相反。 较高的ABA/GA_4比值有利于黄瓜雌性分化,反之则有利于雄性分化。促进黄瓜雄性分化的生长物质GA_3和AgNO_3会引起黄瓜茎端ABA/GA_4比值的下降,而促进其雌性分化的生长物质乙烯利(Ethrel)则会引起ABA/GA_4比值的上升。     

黄瓜植株性别表现与3种氧化酶同工酶的关系

采用同工酶电泳技术分析了二叶期纯雌株和雌雄株黄瓜（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ Ｌ．）子叶和真叶过氧化物酶、多酚氧化酶和超氧化物歧化酶同工酶,结果发现：给株比雌雄株酶活性强、酶带数量多,这种差异酶带大多与雌性或雌雄性别紧密相关,经检验可以作为黄瓜雌性株早期鉴定的生化标记,尤其以真叶中多酚氧化酶同工酶Ｒｆ０．２８７表现稳定,鉴定成功率高。等电聚焦电泳垂直平板聚丙烯酰胺凝胶电泳分辨效果好。     

AFLP marker polymorphism in cucumber (Cucumis sativus L.) near isogenic lines differing in sex expression

The AFLP technique was used to evaluate the level of polymorphism between two pairs of isogenic cucumber (Cucumis sativus L.) lines (NIL) differing in flower sex expression. The BSA techniques were also applied to find molecular markers linked to sex determination genes (dominant alleles) in those cucumber lines. Sex determination in cucumber is controlled by three main loci F, M and Gy. The interaction of these loci is responsible for the formation of the various phenotypes of flowers in respect to sex in the analyzed lines [corrected]. A female line 2gg with a ff/MM/gygy genotype, isogenic to a monoecious line B10 (genotype ff/MM/GyGy), and a female line Gy3 with a FF/MM/GyGy genotype, isogenic to a hermaphroditic line HGy3 (genotype FF/mm/GyGy). Using 56 combinations of AFLP primers, used for the analysis of lines 2gg and B10, gave 3794 bands, of which 155 (4.1%) were polymorphic. Ten bands distinguished gynoecious and monoecious bulks appearing at the same time in the appropriate parent; they are believed to be linked to the Gy locus. The isogenic lines Gy3 and HGy3 showed a higher level of polymorphism (14.2%). In this case, 55 combinations of primers gave 2996 reaction products, of which 430 showed variation. Twenty bands occurred in one bulk and in one parent, so they are probably associated with the M locus. Using the AFLP technique, the isogenicity of the lines was evaluated. The level of polymorphism (per pair of primer) between lines 2gg and B10 is 0.072% and is four times lower than that between the Gy3 and HGy3 lines (0.27%). The differences in the isogenicity of the lines can result from the degree of their relatedness, which may reflect the way they were derived.