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Brief Communications |
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
Received for publication August 21, 2001. Accepted for publication January 24, 2002.
ABSTRACT
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 flowers, such as white campion (Silene latifolia Poiret subsp. alba [= Melandrium album (Miller)] Garcke = Lychnis alba Miller = 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 (Monéger, 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). Löve and Löve (1945)
masculinized red campion (S. dioeca = Melandrium dioecum subsp. rubrum) by applying testosterone to bolting female flower shoots but could not repeat this effect with white campion (S. latifolia = 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 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.
MATERIALS AND METHODS
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 x 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 23°C day/22°C 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 23°C during the day and 22°C 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 80x for the formation of stamens.
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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 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, Ag2S2O3-induced 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 Ag2S2O3-treated females was not viable, as determined by uptake of Alexander's stain (Alexander, 1969
; data not shown). Stamens 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.
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; Beyer, 1976
).
|
; 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 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.
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), 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 1-MCP (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.
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 ethylene-biosysthesis inhibitors, and reduced ethylene-biosynthesis in mung bean (Vigna radiata [L.] Wilczek) by 100% at concentrations of 10 µmol/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.
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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 Ag+? If this hypothesis were true, then other heavy metal compounds might also induce stamens to form in female plants. We sprayed Pb+ and Cu+ 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 Pb+ and Cu+ 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 Ag+ could not be explained as a stress response of the plants to heavy metals.
Possible effects of silver ion application
Only Ag+-treated female white campion produced stamens in this study. Neither ethylene-receptor inhibitors, ethylene-biosynthesis inhibitors, nor sodium thiosulfate alone caused this effect. Although Ag+ 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
), Ag+ did not appear to be acting through inhibition of ethylene in this study. Ag+ has long been recognized as an inhibitor of many sulfhydryl enzymes at concentrations as low as 0.1 µmol/L (Snodgrass, Vallee, and Hock, 1960
), inhibiting both plasma membrane and mitochondrial ATPases (Knee, 1992
). The affinity of Ag+ 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 Ag+. Any of these processes could be involved in stimulation of stamen development by Ag+ in white campion.
FOOTNOTES
1 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 (sgrant{at}e-mail.unc.edu
)
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