American Journal of Botany 89(6): 1014–1020. 2002.
BRIEF COMMUNICATION
SILVER
ENHANCES STAMEN DEVELOPMENT IN FEMALE
(SILENE LATIFOLIA [CARYOPHYLLACEAE])1
WHITE CAMPION
THERESA F. LAW, SABINE LEBEL-HARDENACK,
AND
SARAH R. GRANT2
Department of Biology and Curriculum in Molecular Biology and Genetics, 107 Coker Hall, CB 3280, University of North Carolina, Chapel Hill, North Carolina 27599-3280 USA Sex expression in the dioecious plant white campion (Silene latifolia Poiret subsp. alba) appears to be insensitive to exogenous applications of auxins, cytokinins, gibberellic acid, and ethylene; however, silver thiosulfate (Ag2S2O3), an ethylene inhibitor, enhanced stamen development in female white campion. In wild-type females, stamen development is arrested before the microspore mother cells are formed. In contrast, stamens of Ag2S2O3-treated females completed meiosis and produced microspores. Stamen development for these females was incomplete, however, and pollen did not mature. Ag2S2O3 stimulated stamen development to the same extent in asexual white campion mutants that retained a Y chromosome but had lost Y-linked genes needed for early stages of stamen development. Although Ag2S2O3 can inhibit ethylene signaling, the enhancement of stamen development in female white campion cannot be explained as a loss of ethylene response because no other ethylene inhibitor tested (1-methylcyclopropene, trans-cyclooctene, aminoethoxyvinylglycine, and cobalt chloride) caused stamens to develop in female plants. In addition, application of other metal ions could not enhance stamen development. Therefore, the effect we observed on female white campion was specifically caused by silver ions but not by their action on ethylene signaling. Key words:
Caryophyllaceae; Silene latifolia; silver thiosulfate; stamen development.
Sex expression in many monoecious and dioecious plants is regulated by endogenous hormones and, in some species, can be influenced by the exogenous application of hormones or hormone inhibitors. However, there is no hormone that has a consistently masculinizing or feminizing effect on all species. For example, applications of cytokinins feminize mercury (Mercurialis annua L.) (Durand, 1969; Durand and Durand, 1991), hemp (Cannabis sativa L.) and spinach (Spinacia oleracea L.) (Chailakhyan, 1979), but have no apparent effect on other species, such as cucumber (Cucumis sativus L.) and maize (Zea mays L.) (Chailakhyan, 1979). Gibberellins feminize maize, but masculinize hemp, spinach, cucumber, and asparagus (Asparagus officinalis L.) (Lazarte and Garrison, 1980). Auxins feminize hemp and cucumber, masculinize mercury, and indirectly feminize cucumber by raising ethylene levels in the plant (Heslop-Harrison, 1956; Rudich, Halevy, and Kedar, 1972). It seems that different sex-determination mechanisms involve hormones in different ways. Presumably, the effects of plant hormones on flower development have evolved independently, as monoecious and dioecious species have arisen. Still, because the conserved functions of plant hormones on flower development are poorly characterized, analysis of the role of hormones in sex determination of unisexual flowers could help elucidate the function of phytohormones in developing flowers. In some dioecious plant species possessing unisexual flowManuscript received 21 August 2001; revision accepted 24 January 2002. The authors thank Dr. E. C. Sisler, North Carolina State University, for the gift of the chemicals 1-methylcyclopropene and trans-cyclooctene, Dr. Hyun Sook Chae (University of North Carolina) for assistance with gas chromatography, and Dr. Richard Moore (University of North Carolina) for critical review of the manuscript. This work was supported by USDA grant NRICGP/ USDA 9701317 and NSF grant MCB 9816864. 2 Author for reprint requests (e-mail:
[email protected]). 1
ers, such as white campion (Silene latifolia Poiret subsp. alba [5 Melandrium album (Miller)] Garcke 5 Lychnis alba Miller 5 S. alba [Miller] E. H. L. Krause), sex determination appears to be rigidly controlled by genetics and insensitive to sex conversion by exogenous hormones. In normal female white campion, which possess an XX sex-chromosome complement, stamen primordia are present, but anthers are arrested in development before microspore mother cells or tapetal cells are formed. This occurs because females have male-sterility mutations. These are compensated by dominant stamen-maturation loci on the Y chromosome, which is only present in male (XY) plants (Mone´ger, Barbacar, and Negrutiu, 2000). Ruddat et al. (1991) and Heslop-Harrison (1963) applied steroids, gibberellins, cytokinins, auxins, abscissic acid, and ethylene to flowers of white campion with no apparent effect on either male or female sex expression. We also applied gibberellins and ethylene to male and female white campion with no effect on sex expression (unpublished data). Lo¨ve and Lo¨ve (1945) masculinized red campion (S. dioeca 5 Melandrium dioecum subsp. rubrum) by applying testosterone to bolting female flower shoots but could not repeat this effect with white campion (S. latifolia 5 M. dioecum subsp. album). It remains possible that hormones are important to the sex determination mechanism of white campion, but that it is difficult to produce effective alterations in hormone levels by simply applying excess hormones. Could limiting hormone signaling in planta affect sex expression? In preliminary experiments, we applied inhibitors of gibberellic acid (trinexapac-ethyl and paclobutrazol), an inhibitor of estrogen (tamoxifen), and inhibitors of ethylene (silver nitrate [AgNO3], silver thiosulfate [Ag2S2O3], and aminoethoxyvinylglycine [AVG]) to leaves and flowers of female white campion. Only AgNO3 and Ag2S2O3 caused an effect. When emerging flower shoots
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were exposed to the silver compounds, the flowers that opened subsequently were partially masculinized. The phenotypes of mature female flowers or immature and mature male flowers were not changed by the silver treatments. Because ethylene levels are known to regulate a variety of developmental responses in plants, including sex determination (Johnson and Ecker, 1998), we further investigated the effects of applying ethylene-reducing chemicals to white campion in this study.
TABLE 1. Chemicals selected and Concentrations of chemicals applied to vegetative tissue of female white campion (Silene latifolia) plants. Chemicala
Ethylene receptor inhibitors Silver thiosulfate (Ag2S2O3)
MATERIALS AND METHODS 1-Methylclyclopropene (1-MCP) Plant material—Flowering female white campion inbred families, U9 (Utrect 9, a gift from J. van Brederode, University of Utrecht, The Netherlands), and Duke 1 (15 3 10, a gift from J. Antonovics, Virginia Polytechnic and State University, Virginia, USA), were used in these studies. Preliminary experiments were conducted on both U9 and Duke 1 with similar results. Subsequent experiments were conducted on Duke 1 because plant material was more readily available. Replicate plants were planted in Premier Pro-Mix BX (Premier Horticulture, Dorval, Quebec, Canada), supplemented weekly with Peter’s Special fertilizer (20-20-20 [1 teaspoon/gallon]; Scotts-Sierra Horticultural, Ohio, USA), and were placed in a greenhouse at 238C day/228C night with 15 h light. Plants were allowed to flower prior to the experiments in order to determine the sex. Chemical application—Ethylene-receptor inhibitors Ag2S2O3 (silver nitrate : sodium thiosulfate [Sigma, St. Louis, Missouri, USA], in a 1 : 4 molar ratio [Le Masson and Nowak, 1981]), 1-methylcyclopropene (1-MCP; Serek, Sisler, and Reid, 1994) and trans-cyclooctene (Sisler, Blakenship, and Guest, 1990), ethylene-biosynthesis inhibitors AVG (aminoethoxyvinylglycine) and cobalt chloride (CoCl2; Yu and Yang, 1979), and heavy metal compounds cupric nitrate (Cu[NO3]2) and lead nitrate (Pb[NO3]2) were applied to plants at the concentrations listed in Table 1. Solutions of Ag2S2O3, AVG, CoCl2, Cu(NO3)2, and Pb(NO3)2 were applied as foliar sprays two times, 7 d apart. The 1-MCP and trans-cyclooctene were introduced as gasses at part per billion (PPB) or part per million (PPM) concentrations, respectively, into airtight teflon gas-sampling bags every 2 d for 10 d. After chemical applications, plants were placed into a greenhouse with 15–16 h light at 238C during the day and 228C at night. Seven to ten days after completion of the chemical treatments, each maturing flower bud (5–10 per plant, 2–3 replicate female plants per treatment) was dissected and evaluated at 803 for the formation of stamens. Experiments were in a randomized complete block design, and results were consistent over three replications. Ethylene evolution—Ethylene evolution for the AVG treatments was analyzed for 2–3 replicate flower samples per plant, 2–3 plants per treatment using a Perkin-Elmer HS-40 auto-sampler headspace gas chromatograph equipped with a ParaPLOT U fused silica column (Chrompack from Varian, Palo Alto, California, USA), and the experiment was repeated with similar results.
RESULTS AND DISCUSSION Ag2S2O3 masculinizes female white campion—For all chemical experiments in this study, inflorescences with mature flowers or flower buds were removed prior to chemical treatments to allow only vegetative leaves and leaves of emerging floral shoots to be exposed to the selected chemicals. At this stage, all meristems are vegetative, producing only leaf primoridia (Grant, Hunkirchen, and Saedler, 1994). In preliminary studies, we applied two silver compounds, AgNO3 and Ag2S2O3, to male and female white campion. Both compounds affected female flowers. Female flowers that opened following applications of the silver compounds were partially masculinized. However, AgNO3 was much more phytotoxic than Ag2S2O3, as has been shown in other studies (Le Masson and
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Ethylene biosynthesis inhibitors Trans-cyclooctene
Aminoethoxyvinylglycine (AVG) Cobalt chloride (CoCl2)
Heavy metals Cupric nitrate (Cu[NO3]2)
Lead nitrate (Pb[NO3]2)
Concentration of applied chemicalsb
0 25 50 75 0 10 1000 2000
mmol/L mmol/L mmol/L mmol/L PPB PPB PPB PPB
0 30 60 0 100 0 25 100
PPM PPM PPM mmol/L mmol/L mmol/L mmol/L mmol/L
1 5 10 25 50 1 5 10
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
a Ag S O (1 : 4; volume per volume of equal molar concentrations 2 2 3 of silver nitrate and sodium thiosulfate). CoCl2, Cu[NO3]2, and Pb[NO3]2 were obtained from Sigma (St. Louis, Missouri, USA). 1-MCP and trans-cyclooctene were synthesized and donated by Dr. E. Sisler (North Carolina State University, Raleigh, North Carolina, USA). b Molar solutions of chemicals were sprayed onto plants until run-off two times, 7 d apart, except for 1-MCP and trans-cyclooctene which were introduced as gasses into teflon gas-sampling bags every 2 d for 10 d at parts per million (PPM) and parts per billion (PPB) concentrations.
Nowak, 1981; Veen, 1983), therefore, only Ag2S2O3 was used in subsequent experiments. Application of Ag2S2O3 had no effect on the development of male flowers; however, female flowers produced stamens that were more developed, with longer filaments and larger anther locules (Fig. 1A, B). No other compound tested duplicated this result, and it is important to note that control plants treated with sodium thiosulfate alone applied at the same molar concentrations as the Ag2S2O3 treatments did not form stamens and appeared normal (data not shown). Ag2S2O3-treated female stamens were not as developed as those of normal male flowers (Fig. 1F), and as female flowers matured, Ag2S2O3induced stamens did not develop into mature stamens (Fig. 1C). Nevertheless, anthers of treated female plants remained markedly more developed than in mature untreated female flowers (Fig. 1D). Microspore mother cells, tapetal cells, and pollen were observed in cross-sections of the Ag2S2O3-treated female anthers (Fig. 2D, F), similar to those seen in normal male anthers (Fig. 2B, C); however, pollen from Ag2S2O3treated females was not viable, as determined by uptake of Alexander’s stain (Alexander, 1969; data not shown). Stamens
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TABLE 2. Percentage of female flowers of white campion with stamens following treatment with silver thiosulfate. Chemical
Silver thiosulfate (Ag2S2O3)a
Concentration
0 25 50 75
mmol/L mmol/L mmol/L mmol/L
Flowers with stamens (%)b
0x 12.5x 83.4y 83.0y
a Ag S O (1 : 4, volume per volume of equal molar concentrations of 2 2 3 silver nitrate and sodium thiosulfate) was sprayed onto vegetative leaves and immature flowering shoots until run-off two times, 7 d apart. b Presence of anthers determined at 80 3 in 10–20 female flowers 14–21 d after the first spray with Ag2S2O3. Means with the same letter are not significantly different by Waller-Duncan K ratio t test. K 5 100, P 5 0.05, df 5 19.
of male flowers treated with Ag2S2O3 (Fig. 1E) did not differ morphologically from untreated male stamens (Fig. 1F). Pollen of Ag2S2O3-treated male anthers (Fig. 2E) appeared normal when compared to pollen of untreated male anthers (Fig. 2B) and remained viable as determined by Alexander’s stain. Timing and concentration of Ag2S2O3 affect developmental responses—Both Ag2S2O3 concentration and timing of chemical application were important in inducing stamen formation in female flowers. Stamens were not formed at Ag2S2O3 concentrations below 25 mmol/L, and a significantly higher percentage of stamens formed when the Ag2S2O3 concentration was increased from 25 to 50 mmol/L (Table 2). Ag2S2O3 applications at 75 or 100 mmol/L did not result in a significantly higher number of flowers with stamens, nor were the stamens that formed more morphologically developed than those induced by lower concentrations of Ag2S2O3 (data not shown). Stamens were only observed in female flowers that matured 6–11 d after leaves and flower shoots were sprayed with the second application of Ag2S2O3. Flowers that matured sooner than 6 d or 12–21 d after the final Ag2S2O3 treatment had normal female floral organs. The transient effect of Ag2S2O3 we observed is in agreement with other studies in which the effect of silver ions in cucumber and pea (Pisum sativum L.) plants was of a limited duration (Atsmon and Tabbak, 1979; Beyer, 1976). Does the application of Ag2S2O3 alter stamen development through a common mechanism with Y-linked sex-determination genes?—Three early-stamen-arrest asexual mutants (S68, S34, and S75; Fig. 3A–C) generated by X irradiation (Grant et al., 1994; Donnison et al., 1996) were sprayed with Ag2S2O3 (75 mmol/L) as described above. The Y chromosomes of S34 and S75 have been mapped to locate deletions. Both are missing sequences commonly deleted in mutants with early stamen arrest, but they retain sequences linked to genes ← Fig. 1. Silver thiosulfate (Ag2S2O3, 75 mM) treated and untreated Silene latifolia at petal formation (A, B, E, F) and at mature flower stage (C, D). (A) Ag2S2O3-treated female. (B) Untreated female. (C) Mature Ag2S2O3 treated female. (D) Mature untreated female. (E) Ag2S2O3-treated male. (F) Untreated male. Petals and sepals have been removed to better view sexual structures. Scale bars 5 1mm. Figure Abbreviations: Gyn, gynoecium; Sta, stamen; StaP, stamen primordia; P, pollen; T, tapetum; M, microspore mother cells.
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Fig. 2. Toluidine blue-stained cross-sections through the center of buds of untreated and silver thiosulfate (Ag2S2O3, 75 mM) treated Silene latifolia at petal formation stage (A, B, D, E) and at early anther development (C, F). (A, D) Untreated and Ag2S2O3-treated female, respectively. (B, E) Untreated and Ag2S2O3treated male, respectively. (C, F) Untreated male anther and Ag2S2O3-treated female anther, respectively. Scale bars 5 100 mm.
Fig. 3. Silver thiosulfate (Ag2S2O3, 75 mM) treated and untreated Silene latifolia early stamen-abortion sterile mutants at petal formation stage. (A) Untreated and (D) Ag2S2O3-treated sterile mutant S-68. (B) Untreated and (E) Ag2S2O3-treated sterile mutant S-34. (C) Untreated and (F) Ag2S2O3-treated sterile mutant S-75. Petals and sepals were removed to better view stamens. Scale bars 5 1 mm.
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Fig. 4. Toluidine blue-stained cross-sections of silver thiosulfate (Ag2S2O3, 75 mM) treated and untreated Silene latifolia sterile mutants. (A) Untreated and (C) treated mutant S-34. (B) Untreated and (D) Ag2S2O3-treated mutant S-75. Note that sections were chosen to best show cross-sections through stamen primordia. Scale bars 5 1 mm.
for later stages of stamen development (Lebel-Hardenack et al., 2002). If Ag2S2O3 could mimic the action of the early sex determining genes, then stamens of Ag2S2O3-treated asexual mutants might develop to maturity. When leaves of replicate asexual mutants were sprayed with Ag2S2O3, most flowers (67–100%) formed anthers that were markedly more developed than in untreated mutant flowers (Fig. 3D–F). However, the effect of Ag2S2O3 was not uniform across all stamens within a bud. In most replicate flowers, some of the stamen filaments were not as elongated, and anther locules were not as large as on other stamens within the bud (Fig. 3D–F). Toluidine-stained cross sections of Ag2S2O3-treated mutants showed that the treated stamens developed anther locules and tapetal cells (Fig. 4C, D), but the pollen that developed was not viable, as determined using Alexander’s stain, indicating that Ag2S2O3-enhanced stamen development was still arrested at the same stage as in treated females. Because stamen development was arrested at the same developmental stage in asexual mutants and females, we could not demonstrate that the Ag2S2O3 effect mimics the action of the early sex-determining genes. It is possible that Ag2S2O3 stimulates anther development by a parallel pathway that is independent of the Y-linked sex-determination mechanism. The effect of Ag2S2O3 cannot be explained as inhibition of ethylene signaling—It is important to note that although Ag2S2O3, a known ethylene inhibitor (Beyer, 1976), had a
strong masculinizing effect on female white campion, no other ethylene-receptor or ethylene-biosynthesis inhibitor we applied (Table 1) duplicated this effect. This result was unexpected, indicating that either the stamen-promoting effect we saw with Ag2S2O3 treatments was not caused by inhibition of ethylene signaling or that the other chemicals we tested were actually not active ethylene inhibitors in white campion at the concentrations we applied. To confirm that the ethylene-receptor inhibitor 1-MCP was active in white campion, we introduced 1MCP (2000 PPB) into airtight teflon bags containing female plants, then challenged plants 24 h later with ethylene (100 PPM). Ethylene at 100 PPM caused chlorosis and senescence of foliage and flowers of white campion in preliminary experiments (data not shown). The 1-MCP and the other gaseous ethylene-receptor inhibitor trans-cyclooctene were originally chosen for this study because they are highly effective ethylene inhibitors in a variety of plants even at very low concentrations (0.5 PPB and 0.78 PPM, respectively; Sisler, Blakenship, and Guest, 1990; Sisler, Serek, and Dupille, 1996). In repeated experiments in which we treated plants with 1-MCP and subsequently challenged with ethylene, the plants did not develop chlorotic foliage, even 5–7 d after treatment. In contrast, control plants not treated with 1-MCP developed 70– 90% chlorotic foliage within 48 h of ethylene challenge (data not shown). These results indicated that 1-MCP was an effective ethylene-receptor inhibitor in leaves of white campion at the levels we tested, but could not induce stamens to form.
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TABLE 3. Ethylene evolution in aminoethoxyvinylglycine (AVG)-treated and untreated female white campion flowers. Chemical treatmenta
Ethylene evolution (pL · flower 21 · h21)b
AVG (100 mm) Untreated
76.5 811.0
a Molar solution of AVG was sprayed onto mature female white campion plants two times, 7 d apart. Untreated plants were sprayed with water. b Two to three flowers per plant, three plants per treatment were removed and analyzed 14 d after the initial spray of AVG. Flowers were placed into gas sampling vials 4 h before samples were analyzed. Units are picoliters per flower per hour.
We also did not see stamens develop in female flowers treated with the ethylene-biosynthesis inhibitors AVG and CoCl2. Therefore, we analyzed ethylene evolution by gas chromotography in flowers of plants sprayed with AVG, as described above, to determine if ethylene-biosynthesis was indeed reduced with this treatment relative to untreated control flowers. The AVG and CoCl2 were originally chosen for this study because they have been shown to be highly effective ethylenebiosysthesis inhibitors, and reduced ethylene-biosynthesis in mung bean (Vigna radiata [L.] Wilczek) by 100% at concentrations of 10 mmol/L and 1 mmol/L, respectively (Yu and Yang, 1979). Analysis by gas chromatography showed that untreated mature control flowers produced a tenfold higher level of ethylene than AVG-treated flowers (Table 3), indicating that although AVG appeared to inhibit ethylene-biosynthesis in white campion flowers, it could not induce stamen development in female flowers. Athough they were effective in blocking ethylene perception and synthesis, none of the ethylene inhibitors, other than Ag2S2O3, induced stamen development in female white campion. Therefore, the effect of Ag2S2O3 on female stamen development could not be explained by inhibition of ethylene signaling. Other metal ions or abiotic agents do not enhance stamen development—Plants treated with Ag2S2O3 in this study possessed leaves that were chlorotic, necrotic, and senesced relative to untreated control plants (data not shown). If the ethylene pathway is not involved in stamen production in white campion, are stamens formed in Ag2S2O3-treated female white campion as a result of physical stress caused by application of Ag1? If this hypothesis were true, then other heavy metal compounds might also induce stamens to form in female plants. We sprayed Pb1 and Cu1 compounds at molar concentrations listed in Table 1, with and without sodium thiosulfate, onto replicate female plants. Copper and lead compounds were chosen as heavy metals because they can cause physical stress on plants, including oxidative damage to membranes (Stohs and Bagchi, 1995). In repeated experiments, no stamens were formed in flowers of female plants. The molar concentrations of Pb1 and Cu1 applied in these studies caused symptoms of physical stress on white campion (chlorosis and necrosis of leaves), and higher molar concentrations of the compounds caused either severe necrosis or death of treated plants, indicating that the stamen effect we observed with Ag1 could not be explained as a stress response of the plants to heavy metals. Possible effects of silver ion application—Only Ag1-treated female white campion produced stamens in this study. Neither
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ethylene-receptor inhibitors, ethylene-biosynthesis inhibitors, nor sodium thiosulfate alone caused this effect. Although Ag1 has been shown to be an ethylene-receptor inhibitor and can shift sex expression from female to male in other plant species, presumably through reducing ethylene action in the plant (Beyer, 1976, 1979; Sarath and Mohanram, 1979; Yin and Quinn, 1995), Ag1 did not appear to be acting through inhibition of ethylene in this study. Ag1 has long been recognized as an inhibitor of many sulfhydryl enzymes at concentrations as low as 0.1 mmol/L (Snodgrass, Vallee, and Hock, 1960), inhibiting both plasma membrane and mitochondrial ATPases (Knee, 1992). The affinity of Ag1 for thiol groups and the importance of these groups for structure and activity of plant enzymes suggests that many physiological plant processes could be sensitive to modification by Ag1. Any of these processes could be involved in stimulation of stamen development by Ag1 in white campion. LITERATURE CITED ALEXANDER, M. P. 1969. Differential staining of aborted and non-aborted pollen. Staining Techniques 44: 117–122. ATSMON, D., AND C. TABBAK. 1979. Comparative effects of gibberellin, silver nitrate and aminoethoxyvinyl glycine on sexual tendency and ethylene evolution in the cucumber plant (Cucumis sativus L.). Plant and Cell Physiology 20: 1547–1555. BEYER, E., JR. 1976. Silver ion: a potent antiethylene agent in cucumber and tomato. HortScience 11: 195–196. BEYER, E. M., JR. 1979. Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism. Plant Physiology 63: 169–173. CHAILAKHYAN, M. K. H. 1979. Genetic and hormonal regulation of growth, flowering, and sex expression on plants. American Journal of Botany 66: 717–736. DONNISON, I. S., J. SIROKY, B. VYSKOT, H. SAEDLER, AND S. R. GRANT. 1996. Isolation of Y chromosome-specific sequences from Silene latifolia and mapping of male sex determining genes using representational difference analysis. Genetics 144: 1893–1899. DURAND, B. 1969. Se´lection de ge´notypes males de Mercurialis annua L. (2n 5 16) en fonction de leur sensibilite´ aux cytokinines. Comptes Rendu de L’Academie des Science Paris, se´rie D 268: 2409–2051. DURAND, B., AND R. DURAND. 1991. Male sterility and restored fertility in annual mercuries, relations with sex differentiation. Plant Science 80: 107–118. GRANT, S., A. HOUBEN, B. VYSKOT, J. SIROKY, W.-H. PAN, J. MACAS, AND H. SAEDLER. 1994. Genetics of sex determination in flowering plants. Developmental Genetics 15: 214–230. GRANT, S. R., B. HUNKIRCHEN, AND H. SAEDLER. 1994. Developmental differences between male and female flowers in the dioecious plant Silene latifolia. Plant Journal 6: 471–480. HESLOP-HARRISON, J. 1956. Auxin and sexuality in Cannabis sativa. Physiological Planta 4: 588–597. HESLOP-HARRISON, J. 1963. Sex expression in flowering plants. Brookhaven Symposium of Quantitative Biology 16: 109–125. JOHNSON, P. R., AND J. R. ECKER. 1998. The ethylene gas signal transduction pathway: a molecular perspective. Annual Review of Genetics 32: 227– 254. KNEE, M. 1992. Sensitivity of ATPases to silver ions suggests that silver acts outside the plasma membrane to block ethylene action. Phytochemistry 31: 1093–1096. LAZARTE, J. E., AND A. GARRISON. 1980. Sex modification in Asparagus officinalis L. Journal of the American Society for Horticultural Science 105: 691–694. LEBEL-HARDENACK, S., E. HAUSER, T. F. LAW, J. SCHMID, AND S. R. GRANT. 2002. Mapping of sex determination loci on the white campion (Silene latifolia) Y chromosome using AFLP. Genetics 160: 717–725. LE MASSON, B., AND J. NOWAK. 1981. Cut flower life of dry transported carnations as influenced by different silver form pre-treatments. Scientia Horticulturae 15: 383–390. LO¨VE, A., AND D. LO¨VE. 1945. Experiments on the effects of animal sex hormones on dioecious plants. Arkiv fo¨r Botanik 13: 1–60.
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