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; )     

The Interaction of Genetic, Environmental, and Hormonal Factors in Stem Elongation and Floral Development of Cucumber Plants

Hypocotyl length was found to vary between cucumber plants carryingdifferent genes controlling sex expression. Among lines havingonly unisexual flowers (genotype M/M), the homozygous monoeciousplants (st⁺/st⁺) had significantly longer hypocotyls than theirgynoecious counterparts (st/st), heterozygous gynoecious plants(st+/st) being intermediate. Similarly, hypocotyls of plantsof an andromonoecious line (st⁺/st⁺ m/m) were significantlylonger than in their hermaphrodite counterparts (st/st m/m).Differences in intemode length were also significant and inthe same direction. Since stem and particularly hypocotyl elongationin cucumber is known to be very sensitive to applied gibberellin,these findings suggest the existence of differences in the effectivelevels of endogenous gibberellins in the different sex types,higher levels being correlated with stronger male tendency.This conclusion is in good agreement with the known effect ofapplied gibberellin on sex expression (enhancement of the maletendency) in cucumber. Application of gibberellin (GA₄+GA₇) and exposure to ‘summer’conditions (long days and relatively high temperature) inhibitedthe development of pistillate flowers while ‘winter’conditions (short days, lower temperature) had a similar effecton staminate flowers. The effect was in either case specific,that is, limited to flower development. It is concluded thatexogenous and endogenous gibberellins affect not only the initiation,but also the further development, of flowers in the cucumber.     

Involvement of abscisic acid in the regulation of sex expression in the cucumber

The interaction between abscisic acid (ABA), gibberellin (GA)and ethephon on sex determination in cucumber (Cucumis sativusL.) flowers was examined. ABA promoted the female tendency ofgynoecious plants, but did not change the sex expression ofmonoecious ones. When ABA was applied together with GA₄₊₇ thepromoting activity of the GA on male flower formation in thegynoecious line was reduced. ABA also inhibited tendril appearanceand internode length, characteristic of GA treatments. A combinedABA and ethephon treatment resulted in a synergistic activityinhibiting growth and increasing the period of female flowerappearance in the monoecious line. It is suggested that ABAparticipates in the sex regulation of the cucumber by inhibitingGA activity. (Received March 8, 1974; )     

Inhibition of Flowering in the Long-Day Plant Lemna gibba G3 by Hutner's Medium and its Reversal by Medium Modification

The long-day plant Lemna gibba G3 flowers normally in E medium(Hoagland-type medium plus 30 µM EDTA) but in 0.5 H mediumthere is no flowering. Ammonium is present in 0.5 H medium andis known to inhibit flowering in L. gibba G3, but even in NH₄⁺-free0.5 H medium there is virtually no flowering under continuouslight. Increasing the phosphate concentration of the NH₄⁺-free0.5 H medium from 1.15 ITIM to 12 or 16 mM results in substantialflowering. Decreasing the EDTA concentration from 850 µIMto 250 µM, or raising the nitrate concentration from 4mM to 12 mM, results in only a small increase in flowering.If the decrease in EDTA and increase in nitrate are combinedwith the increase in phosphate, however, the flowering responseis nearly as good as that obtained using E medium. Thus, withthese three changes the inhibitory effect of NH₄⁺free 0.5 Hmedium for flowering in L. gibba G3 is almost completely reversed In the above studies flowering was not limited by daylength.When plants were grown on E medium under an 11 hour daylengthwhere flowering is limited by daylength, decreasing the phosphateconcentration in the medium reduced flowering, but increasingthe phosphate concentration in the medium did not stimulateflowering. Thus, when flowering is limited by daylength, highphosphate will not cause flowering, but a certain level of phosphateappears to be necessary for the expression of photoinductionunder long days. (Received January 14, 1986; Accepted June 24, 1986)     

Comparative effects of gibberellin, silver nitrate and aminoethoxyvinyl glycine on sexual tendency and ethylene evolution in the cucumber plant (Cucumis sativus L.)

All three experimental compounds induced, after two sprays,the development of staminate flowers in otherwise strictly gynoeciouscucumbers. Silver nitrate and aminoethoxyvinyl glycine (AVG)also induced perfect flowers and the appearance of multiplepistillate and/or perfect buds on each node. Gibberellin (A₄₊₇) had no effect on ethylene evolution, silvernitrate increased it, and AVG drastically reduced it. Thesedifferences between the three compounds indicate different mechanismsfor the male-enhancing effects. Inhibition of ethylene biosynthesisby AVG and inhibition of its action and/or metabolism by silvernitrate, leading to a decrease in endogenous ethylene activityand thus to an enhanced male tendency. The GA effect does notseem to involve a change in ethylene content, and thereforemust be through another, yet unknown, mechanism. (Received August 9, 1979; )     

Behavior of current-year shoots as a mechanism to determine the floral sex allocation at the level of individual tree and population in Siberian alder (Alnus hirsuta var. sibirica)

Floral sex allocation (weight of male flower buds over weight of female flower buds) was examined at the levels of current-year shoot, individual tree and population, and the tree individual level and population level floral sex ratio was explained as a consequence of the behavior of current-year shoots in the shoot-level monoecious (flowering current-year shoots have both male and female flowers) species, Siberian alder (Alnus hirsuta var. sibirica). The current-year shoot level floral sex allocation was not size-dependent and not different over years. However, in the year when the reproductive intensity was high, individual tree level floral sex allocation was size-dependent and the population level floral sex allocation was relatively female-biased. The female-biased floral sex allocation at the population level resulted from many gynoecious shoots (current-year shoots which have only female flowers). These results suggest that the floral sex allocation of Siberian alder was controlled not by changing the floral sex allocation of each current-year shoot, but by shifting the sex expression of current-year shoots from shoot-level monoecy to shoot-level gynomonoecy.     

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; )     

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.     

Modelling of the Partitioning, Assimilation and Storage of Nitrate within Root and Shoot Organs of Castor Bean (Ricinus communis L.)

An experimentally-based modelling technique was developed todescribe quantitatively the uptake, flow, storage and utilizationof NO₃-N over a 9 d period in mid-vegetative growth of sandcultured castor bean (Ricinus communis L.) fed 12 mol m^–3nitrate and exposed to a mean salinity stress of 128 mol m^–3NaCl. Model construction used information on increments or lossesof NO₃-N or total reduced N in plant parts over the study periodand concentration data for NO₃-N and reduced (amino acid) Nin phloem sap and pressure-induced xylem exudates obtained fromstem, petiole and leaf lamina tissue at various levels up ashoot. The resulting models indicated that the bulk (87%) of incomingnitrate was reduced, 51% of this in the root, the remainderprincipally in the laminae of leaves. The shoot was 60% autotrophicfor N through its own nitrate assimilation, but was oversuppliedwith surplus reduced N generated by the root and fed to theshoot through the xylem. The equivalent of over half (53%) ofthis N returned to the root as phloem translocate and, mostly,then cycled back to the shoot via xylem. Nitrate comprised almosthalf of the N of most xylem samples, but less than 1% of phloemsap N. Laminae of leaves of different age varied greatly inN balance. The fully grown lower three leaves generated a surplusof reduced N by nitrate assimilation and this, accompanied byreduced N cycling by xylem to phloem exchange, was exportedfrom the leaf. Leaf 4 was gauged to be just self-sufficientin terms of nitrate reduction, while also cycling reduced N.The three upper leaves (5–7) met their N balance to varyingextents by xylem import, phloem import (leaves 6 and 7 only)and assimilation of nitrate. Petioles and stem tissue generallyshowed low reductase activities, but obtained most of theirN by abstraction from xylem and phloem streams. The models predictedthat nodal tissue of lower parts of the stem abstracted reducedN from the departing leaf traces and transferred this, but notnitrate, to xylem streams passing further up the shoot. As aresult, xylem sap was predicted to become more concentratedin N as it passed up the shoot, and to decrease the ratio ofNO₃-N to reduced N from 0·45 to 0·21 from thebase to the top of the shoot. These changes were reflected inthe measured N values for pressure-induced xylem exudates fromdifferent sites on the shoot. Transfer cells, observed in thexylem of leaf traces exiting from nodal tissue, were suggestedto be involved in the abstraction process. Key words: Ricinus communis, nitrogen, nitrate, nitrate reduction, partitioning, phloem, xylem, flow models     

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.     

Suppression of long-day flowering by nitrogenous compounds in Lemna perpusilla 6746

The long-day flowering of Lemna perpusilla 6746 on an SH inhibitor-containingmedium was inhibited by the application of ammonium ion to themedium. Ammonium ion not only suppressed long-day flowering,but relieved the inhibition of vegetative growth caused by theinhibitors. Nitrite, casamino acids, glutamine and asparaginehad a similar effect, suggesting that the inhibition of long-dayflowering by ammonium ion is not a direct effect of the ion.Most amino acids, with the exception of glutamate and aspartate,also prevented long-day flowering, but their effects on vegetativegrowth varied. No qualitative differences in amino acid compositionwere observed among plants cultured on media containing nitrate,nitrite or NH4₄NO₃as the sole nitrogen source. However, theamounts of free and total amino acids werehigher in plants fedwith nitrite or NH₄NO₃ than in those fed with nitrate. Thissuggests that the inhibition of long-day flowering by ammoniumand nitrite can be ascribed to increased nitrogen metabolism. Though decreased activity by SH inhibitors of nitrate reductase(SH enzyme) is assumed to result in long-day flowering by loweringthe nitrogen metabolism, lowering the nitrogen level in M mediumdid not bring about floral initiation in the absence of SH inhibitors. (Received January 7, 1975; )     

Role of Ethylene in Senescence of Petals--Morphological and Taxonomical Relationships

Petal senescence in mature flowers was studied in 93 speciesfrom 22 families. The initial symptom of senescence was eitherwilting or abscission, but in some species the time span betweenwilting and abscission was very short. There was no apparent relationship between corolla form (choripetalousor sympetalous), ovary position (inferior or superior with respectto the corolla) and type of senescence (initial wilting or initialabscission). In monocots no initial abscission was found, whilein dicots the difference between the wilting type and the abscissiontype was generally at the family level. With respect to petalsenescence, sensitivity to exogenous ethylene (C₂H₄) was alsorelated to the family level. Except for a few families (all tested Campanulaceae, Caryophyllaceaeand Malvaceae, and most Orchidaceae), most of the flowers investigatedthat showed initial wilting were not sensitive to exogenousethylene, e.g. all tested Compositae, Iridaceae, and Liliaceae.Most of the flowers showing initial abscission were sensitiveto exogenous ethylene (Geraniaceae, Labiatae, Ranunculaceae,Rosaceae, Scrophulariaceae). Experiments with silver thiosulphate (STS) confirmed the effectsof exogenous ethylene, both in flowers showing initial wiltingand in flowers showing initial abscission. The data indicate,therefore, that ethylene is involved in the natural senescenceof only a minority of the wilting type of flowers and in a majority(if not all) of the abscising type of flowers. Key words: Abscission, ethylene, senescence, silver thiosulphate     

Sexual differentiation in cucumber: Abscisic acid and gibberellic acid contents of various sex genotypes

The AbA content of cucumber shoot tips was determined by GLC.Shoot tips of monoecious plants had a higher AbA content thanthose of gynoecious ones, and SD conditions caused higher AbAcontent than LD conditions. AbA content per unit dry weightof young floral buds was 5- to 9-fold higher than in older ones. A GLC method was developed to allow parallel identificationand quantitative determinations of GA₃ and AbA with the samesample. The shoot tip contents of both growth substances weredetermined at four developmental stages of the monoecious plant.Both significantly increased until the 12th day after plantingthen decreased; the GA₃ content decreased markedly. A general hypothesis for the role of growth substances in regulatingsex expression of cucumber is presented and discussed. (Received January 7, 1977; )     

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.     

Effects of nitrogen nutrition on salt-stressed Nicotiana tabacum var. Samsun in vitro plantlets

Tobacco shoots were grown in vitro for 35 d, in MS culture mediummodified to include various sources (nitrate-N, ammonium-N ora mixture) and levels (0–120 mM) of N, and in the presenceof 0–180 mM NaCI or iso-osmotic concentrations of mannitol.Growth of control plantlets was significantly inhibited whenNH₄⁺-N was the sole N source, and at high (120 mM) NO^–₃-N supply. Under conditions of salt stress (90 and 180 mM NaCI)growth was repressed, with roots being more severely affectedthan shoots. Salinity also inhibited root emergence in vitro.The only alleviation of the salt stress by nitrate nutritionobserved in this study was on shoot growth parameters of plantletsgrown on 60 mM NO^–₃-N and 90 mM NaCI. Although both weresignificantly inhibited by NaCI, nitrate reduc-tase activitywas more severely affected than nitrate uptake. When mannitolreplaced NaCI in the culture medium, similar Inhibition of growth,nutrient uptake and enzyme activity were recorded. These observations,together with the relatively low recorded values for Na⁺ andCI^– uptake, indicate that under in vitro salt stress conditionsthe negative effects of NaCI are primarily osmotic. Key words: Growth, nitrogen metabolism, osmotic stress, salinity     

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.     

Purification and properties of a nitrate reductase inactivating factor from rice cells in suspension culture

The nitrate reductase inactivating factor in cultured rice cellswas purified 320-fold. The purification procedure involved precipitationwith (NH₄)₂SO₄, fractionation at pH 4.0, adsorption on CM-cellulose,and gel filtration on Sephadex G-200. The molecular weight wasestimated to be 200,000 from the Sephadex G-200 gel filtration. The inactivating factor shows maximal activity at pH 8.0 andappears to be located in the cytoplasm of the cultured ricecells. The inactivating factor was more stable to heat treatmentthan NADH nitrate reductase. The factor inactivated nitratereductase complex except for reduced methylviologen nitratereductase. It had no influence on the activity of nitrite reductase,glutamate dehydrogenase, and NADH diaphorase, but inactivatedxanthine oxidase. The inactivating factor had no protease activitywhen casein, bovine serum albumin, or nitrate reductase fractionwas used as the substrate. The type of inactivation of nitratereductase by the inactivating factor was noncompetitive. Inhibitionof the inactivating factor by o-phenanthroline, EDTA, and p-chloromercuribenzoicacid suggested the involvement of a metal and sulfhydryl groupat its active site. (Received January 28, 1977; )