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.     

Sexual differentiation in Asparagus officinalis L.

Summary Using an immunological method we assayed the levels of auxin, abscisic acid and three cytokinins (transzeatin riboside, dihydrozeatin riboside, isopentenyladenosine) in flowers of female and male plants of Asparagus officinalis L. at different stages of development. The largest differences between the sexes were found for auxin: auxin content was found to be about three times higher in young male flowers than in female flowers at a corresponding developmental stage. In order to identify some of the biochemical markers linked to sex differentiation, we also examined peroxidase isoenzyme patterns during flower development. We found five flower-specific peroxidase bands, three of which appear to be localized in the anthers. In young flowers still sexually undifferentiated in their morphology these bands are present in both sexes. They subsequently rapidly disappear in the female flower (approximately at the same time as when anther development is blocked), while they persist for a much longer time in the male. The temporary presence of these peroxidase isoenzymes in female young flowers together with the large difference in auxin content indicate that the stage of the young flower is a crucial moment in the process of sex determination.     

栝楼不同性别花芽分化形态解剖特征观察

采用体视显微镜、石蜡切片和树脂切片技术对栝楼(Trichosanthes kirilowii Maxim.)不同性别花芽分化发育时期的外部形态和内部解剖结构进行了观察。结果显示,栝楼花为雌雄异株,仅有雌花、雄花两种性别分化,且雄花的发育速度明显快于雌花的发育速度。栝楼雌雄花芽长0.2 mm左右已完成性别分化;栝楼雄花为单性花,分化过程可分为6个时期,整个发育过程仅见雄蕊原基的分化及生长。栝楼雌花为"两性花",分化过程可分为7个时期,存在雌蕊和雄蕊共同发育阶段,后期雄蕊发育败退。本研究明确了不同性别栝楼花芽发育发生的各个阶段、形态变化特点、外部形态变化特征以及雌雄花芽的分化差异,建立了雌雄花芽内部结构分化与外部形态之间相关性,为栝楼早期幼苗鉴定及性别分化研究提供了一定的参考。     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

麻疯树雌雄花中MADS-BOX基因的表达分析

麻疯树(Jatropha curcas)种子含油率高,种子中的油类物质可作为生物柴油被开发和利用,是极具潜力的生物质能源树种之一。麻疯树雌雄异花,在自然条件下雄花数量通常远远大于雌花,这大大限制了种子和油的产量,因此开展麻疯树性别分化与花发育分子机理的研究具有重要意义。该研究选取10个麻疯树的MADS-BOX基因(JcAGL1,JcAGL6,JcAGL9,JcAGL11,JcAGL15,JcAGL61-3,JcAGL62-1,JcAGL62-6,JcAGL62-7,JcAGL80-2),提取麻疯树早期发育各个阶段的雌雄花总RNA,并反转录成cDNA,采用实时荧光定量方法,探索早期发育不同阶段的麻疯树雌雄花目的基因的表达情况。结果表明:目的基因在发育起始的雌雄花中的表达具有差异,比如JcAGL6和JcAGL15在雄花中表达量要高于雌花,而JcAGL1,JcAGL9和JcAGL11在雌花中的表达量要高于雄花,这说明花原基中目的基因表达会直接或间接决定性别分化的方向;在之后的发育过程中,目的基因的表达情况在雌雄花中有所不同:随着花的发育,目的基因在雌雄花中的表达量变化存在差别,这反应出麻疯树雌雄花发育中目的基因表达模式上的差异;另外,也能看出在此过程中各个目的基因又发挥着不同的功能。该研究结果为进一步探究麻疯树雌雄花发育相关基因的表达提供了理论依据,为了解麻疯树性别分化和花发育的分子机理奠定了基础。     

Inception of maleness: auxin contribution to flower masculinization in the dioecious cactus Opuntia stenopetala

In Opuntia stenopetala, flowers initiate as hermaphrodite; however, at maturity, only the stamens in male flowers and the gynoecium in female flowers become functional. At early developmental stages, growth and morphogenesis of the gynoecium in male flowers cease, forming a short style lacking stigmatic tissue at maturity. Here, an analysis of the masculinization process of this species and its relationship with auxin metabolism during gynoecium morphogenesis is presented. Histological analysis and scanning electron microscopy were performed; auxin levels were immunoanalyzed and exogenous auxin was applied to developing gynoecia. Male flower style-tissue patterning revealed morphological defects in the vascular bundles, stylar canal, and transmitting tissue. These features are similar to those observed in Arabidopsis thaliana mutant plants affected in auxin transport, metabolism, or signaling. Notably, when comparing auxin levels between male and female gynoecia from O. stenopetala at an early developmental stage, we found that they were particularly low in the male gynoecium. Consequently, exogenous auxin application on male gynoecia partially restored the defects of gynoecium development. We therefore hypothesize that, the arrest in male flower gynoecia patterning could be related to altered auxin homeostasis; alternatively, the addition of auxin could compensate for the lack of another unknown factor affecting male flower gynoecium development.     

The Control of Sex Expression in Cucurbits by Ethephon

Application of ethephon to field-grown plants of both bush andtrailing forms of Cucurbita maxima and C. pepo caused leaf epinasty,suppression of male flowers and earlier production and increasein numbers of female flowers. This gave rise to an increasein the ratio of female to male flowers per plant and a decreasein the total number of flowers. Observations of C. pepo showed that even at the two true leafstage there are several nodes present in the unexpanded shoot.Each node has one main and several secondary buds. The sex ofthe main bud at the first five to six nodes is usually determinedat this stage but the secondary buds still have bisexual potential.The change in sex expression was brought about by all male flowerbuds that had formed by the spraying time aborting, and allbuds that developed (both main and secondary) for at least 7days after spraying became female flowers. Thus, nodes fiveand six had male flowers in the controls, whereas in ethephon-sprayedplants the presumptive male flowers aborted at the bud stageat these nodes and secondary primordia developed into functionalfemale flowers. Cucurbita maxima, Cucurbita pepo, sex expression, ethephon, ethylene, flower abortion, flower differentiation     

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.     

Comparative floral development in male and female plants of Palmer amaranth (Amaranthus palmeri)

Premise

Characterizing the developmental processes in the transition from hermaphroditism to unisexuality is crucial for understanding floral evolution. Amaranthus palmeri, one of the most devastating weeds in the United States, is an emerging model system for studying a dioecious breeding system and understanding the biological traits of this invasive weed. The objectives of this study were to characterize phases of flower development in A. palmeri and compare organogenesis of flower development in female and male plants.

Methods

Flower buds from male and female plants were dissected for light microscopy. Segments of male and female inflorescences at different stages of development were cut longitudinally and visualized using scanning electron microscopy.

Results

Pistillate flowers have two to three styles, one ovary with one ovule, and five obtuse tepals. Staminate flowers have five stamens with five acute tepals. Floral development was classified into 10 stages. The distinction between the two flower types became apparent at stage four by the formation of stamen primordia in staminate flowers, which developed female and male reproductive organs initially, as contrasted to pistillate flowers, which produced carpel primordia only. In staminate flowers, the putative carpel primordia changed little in size and remained undeveloped.

Conclusions

Timing of inappropriate organ termination varies across the two sexes in A. palmeri. Our study suggests that the evolution of A. palmeri from a cosexual ancestral state to complete dioecy is still in progress since males exhibited transient hermaphroditism and females produced strictly pistillate flowers.     

慈姑花的开放式样及其花粉流

对一野生慈姑（ＳａｇｉｔａｒｉａｔｒｉｆｏｌｉａＬ．）居群的性表达状态及其花粉流散布规律进行了观测。慈姑雌雄同株,单株雄花数是雌花数的３．６倍。雌雄花单花期均为１ｄ,但二者开放式样不同。尽管雌花的数目较少,但在１ｄ内开放较多的花,其花的开放速率比雄花快。在居群水平上表现出雌雄花比例随时间大幅度波动。在传粉者进行的２６１２次访花活动中,传粉者表现出偏“雌性”访问,对每朵雌花的访问是雄花的１．４倍。用番红染色法标记慈姑的花粉运动表明,花的不同开放式样影响花粉流的散布。当雄花数占高比例时,花粉主要在邻近的个体间散布;而雄花的比例较低、雌花相对较多时,花粉远距离散布的强度加大。     

The arrest of development of abortive reproductive organs in the unisexual flower of<Emphasis Type="Italic"> Vitis vinifera</Emphasis> ssp.<Emphasis Type="Italic"> silvestris</Emphasis>

During the first stages of development, flowers of most dioecious species are hermaphroditic, with their transition to unisexual flowers being the result of the developmental arrest of one set of reproductive organs. In this work, we describe the development of male and female flowers of the dioecious wild grape species Vitis vinifera ssp. silvestris through scanning electron microscopy analysis and cytological observations, focusing our attention on the transition from bisexual to unisexual development. We divide floral development of the wild grape into eight stages. Differences between male and female flowers appear first at stage 6, when the style and stigma start to differentiate in female but not in male flowers. Cytological analysis of the slowly growing abortive pistil of male flowers shows that megagametophyte formation is, surprisingly, not inhibited. Instead of pistil abortion in the male flower, sexual determination is accomplished through programmed death of external nucellus cells and some layers of integumentary cells. Sterility of male structures in female flowers follows a different pattern, with microspore abnormalities evident from the time of their release from the tetrad. Sterile microspores and pollen grains in female flowers display an abnormal round shape, lacking colpi and possessing uniformly thickened cell walls that impede germination.     

Sex-Biased Temporal Gene Expression in Male and Female Floral Buds of Seabuckthorn (Hippophae rhamnoides)

Seabuckthorn is an economically important dioecious plant in which mechanism of sex determination is unknown. The study was conducted to identify seabuckthorn homologous genes involved in floral development which may have role in sex determination. Forty four putative Genes involved in sex determination (GISD) reported in model plants were shortlisted from literature survey, and twenty nine seabuckthorn homologous sequences were identified from available seabuckthorn genomic resources. Of these, 21 genes were found to differentially express in either male or female flower bud stages. HrCRY2 was significantly expressed in female flower buds only while HrCO had significant expression in male flowers only. Among the three male and female floral development stages (FDS), male stage II had significant expression of most of the GISD. Information on these sex-specific expressed genes will help in elucidating sex determination mechanism in seabuckthorn.     

Characterization of unisexual flower development in the endangered mahogany tree Swietenia macrophylla King. (Meliaceae)

The selection of candidate plus trees of desirable phenotypes from tropical forest trees and the rapid devastation of the natural environments in which these trees are found have created the need for a more detailed knowledge of the floral and reproductive biology of tropical tree species. In this article, the organogenic processes related to unisexual flower development in tropical mahogany, Swietenia macrophylla , are described. Mahogany inflorescences at different developmental stages were evaluated using scanning electron microscopy or optical microscopy of histological sections. The unisexual flowers of S. macrophylla are usually formed in a thyrse, in which the positions of the female and male flowers are not random. Differences between male and female flowers arise late during development. Both female and male flowers can only be structurally distinguished after stage 9, where ovule primordia development is arrested in male flowers and microspore development is aborted in female flower anthers. After this stage, male and female flowers can be distinguished by the naked eye as a result of differences in the dimensions of the gynoecium. The floral characteristics of S. macrophylla (distribution of male and female flowers within the inflorescence, and the relative number of male to female flowers) have practical implications for conservation strategies of this endangered species.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 529–535.     

Comparative ontogeny of the cyathium in Euphorbia (Euphorbiaceae) and its allies: exploring the organ-flower-inflorescence boundary

We present a detailed comparative ontogenetic analysis of pseudanthia of representatives of all three subtribes of Euphorbieae (Euphorbiinae, Neoguillauminiinae, Anthosteminae) in order to clarify their homologies and interpretation. The cyathium of Euphorbia and its allies (subtribe Euphorbiinae) closely resembles a bisexual flower but is traditionally interpreted as an inflorescence bearing clusters of highly reduced male flowers surrounding a single terminal female flower. Previously unreported characters are (1) male flowers formed one above the other in the male inflorescences of some Euphorbiinae, (2) late-developing perianthlike structures in some male flowers of Neoguillauminia cleopatra, (3) evidence for a bracteate origin of the female perianth in Anthosteminae and Neoguillauminiinae, and (4) spatiotemporally independent formation of abscission zone and perianth. Indistinct boundaries between inflorescence, flower, and floral organs demonstrate that defining the cyathium neither as an inflorescence nor as a flower is entirely satisfactory and indicate a "hybrid" flower/inflorescence nature of the cyathium. Based on our current knowledge and the existing phylogenetic context, it is most parsimonious to suggest that the cyathium evolved from a determinate thyrse with a terminal female flower surrounded by dichasial male partial inflorescences. We speculate that the cyathium was formed because of strong condensation and possible overlap between expression zones of regulatory genes.     

Floral structure and development in the monoecious palm Gaussia attenuata (Arecaceae; Arecoideae)

Background and Aims

Sexual dimorphism, at both the flower and plant level, is widespread in the palm family (Arecaceae), in contrast to the situation in angiosperms as a whole. The tribe Chamaedoreeae is of special interest for studies of the evolution of sexual expression since dioecy appears to have evolved independently twice in this group from a monoecious ancestor. In order to understand the underlying evolutionary pathways, it is important to obtain detailed information on flower structure and development in each of the main clades.

Methods

Dissection and light and scanning electron microscopy were performed on developing flowers of Gaussia attenuata, a neotropical species belonging to one of the three monoecious genera of the tribe.

Key Results

Like species of the other monoecious genera of the Chamaedoreeae (namely Hyophorbe and Synechanthus), G. attenuata produces a bisexual flower cluster known as an acervulus, consisting of a row of male flowers with a basal female flower. Whereas the sterile androecium of female flowers terminated its development at an early stage of floral ontogeny, the pistillode of male flowers was large in size but with no recognizable ovule, developing for a longer period of time. Conspicuous nectary differentiation in the pistillode suggested a possible role in pollinator attraction.

Conclusions

Gaussia attenuata displays a number of floral characters that are likely to be ancestral to the tribe, notably the acervulus flower cluster, which is conserved in the other monoecious genera and also (albeit in a unisexual male form) in the dioecious genera (Wendlandiella and a few species of Chamaedorea). Comparison with earlier data from other genera suggests that large nectariferous pistillodes and early arrest in staminode development might also be regarded as ancestral characters in this tribe.     

Unisexual flower, spikelet, and inflorescence development in monoecious/dioecious Bouteloua dimorpha (Poaceae, Chloridoideae)

Unisexual flowers have evolved repeatedly in the angiosperms. In Poaceae, multiple transitions from bisexual to unisexual flowers are hypothesized. There appear to be at least three distinct developmental mechanisms for unisexual flower formation as found in members of three subfamilies (Ehrhartoideae, Panicoideae, Pharoideae). In this study, unisexual flower development is described for the first time in subfamily Chloridoideae, as exemplified by Bouteloua dimorpha. Scanning electron microscopy (SEM) and anatomy were used to characterize the development of male (staminate) and female (pistillate) flowers, spikelets, and inflorescences. We found the developmental pathway for staminate flowers in B. dimorpha to be distinct from that described in the other three subfamilies, showing gynoecial arrest occurs at a different stage with possible loss of some cellular contents. However, pistillate flowers of B. dimorpha had some similarity to those described in other unisexual-flowered grasses, with filament and anther differentiation in abortive stamens. Comparing our findings with previous reports, unisexual flowers seem to have evolved independently in the four examined grass subfamilies. This analysis suggests the action of different genetic mechanisms, which are consistent with previous observations that floral unisexuality is a homoplasious condition in angiosperms.     

心叶凹唇姜的开花格局和雄性先熟机制

心叶凹唇姜 (Boesenbergia longiflora) 是分布于亚洲热带的一种姜科植物, 在我国滇南地区成片生长于石灰山贫瘠的岩石缝隙和路边林下的冲积土壤上。心叶凹唇姜的花寿命为两天, 第1天为严格的雄性阶段, 第2天为严格的雌性阶段, 是典型的花内雄性先熟, 一朵花的雌、雄性阶段没有重叠, 因而完全避免了自花授粉的发生。对这两种生境 (石灰山贫瘠的岩石缝隙和路边林下) 下的心叶凹唇姜的4个野外种群 (在云南省普洱市的翠云乡彩虹洞、南岛河、澜沧县惠民乡和西双版纳州绿石林森林公园) 和栽培植株的开花格局进行调查后发现, 大多数个体在同一时间内只有一个花序开花, 并且每隔一天才开一朵花, 只有10%左右的植株同时具有雄性阶段和雌性阶段的花, 这样的开花格局有效地降低了同株异花授粉发生的可能。心叶凹唇姜的平均花粉-胚珠比 (Pollen-ovuleratio) 为291, 应该是以异交为主的混合交配系统。其自然种群存在着花粉限制, 彩虹洞、南岛河和绿石林3个不同种群的平均结籽率都不到60%, 这可能与其较小的花展示有关。心叶凹唇姜为泛化传粉植物, 不同种群的有效传粉昆虫种类各不相同。在两种不同的生境条件下, 心叶凹唇姜花的大小和开花格局都没有显著的差异, 大多数个体同时只具有一种性阶段的花, 说明心叶凹唇姜这种独特的开花格局是长期进化的结果。心叶凹唇姜通过隔一天开一朵花的开花格局, 配合雄性先熟机制, 有效地降低了自交的发生。