The role of ethylene and brassinosteroids in the control of sex expression and flower development in <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

In this paper we compare the sensitivity of different squash genotypes to ethylene and brassinosteroids by studying the effects of different ethylene and brassinosteroid treatments on the sexual expression and flower development of different C. pepo genotypes: Bolognese (Bog) and Vegetable Spaghetti (Veg), two contrasting lines for ethylene production and sensitivity, as well as Cora, a standard commercial hybrid. Results have demonstrated that ethylene has a much greater effect on sexual expression and flower development in C. pepo than brassinosteroids. Ethephon increases the number of female flowers per plant and reduces the first male phase of development, while treatments with the ethylene inhibitors AVG and STS reduce the number of female flowers per plant and expand the first male phase of development. The differential response observed between genotypes appears to be related to ethylene production and sensitivity. Bog, which produces more ethylene and is more sensitive to this hormone, responded much better to AVG and STS, reducing the number of female flowers per plant, while Veg, which is characterised by lower production of and sensitivity to ethylene, responded better to ethephon by reducing the first male phase of development and increasing the number of female flowers per plant. The differential abortion of female and male flowers in ethephon, AVG and STS treatments, as well as the occurrence of bisexual flowers in the AVG and STS treated plants of the more ethylene sensitive genotypes, demonstrate that ethylene is also involved in the development of female flowers. Female flower buds require a minimal level of ethylene not only to complete their development and maturation without a premature abortion, but also to arrest the development of stamens in the third whorl and to promote the appropriate growth of the carpels. On the contrary, the role of brassinosteroids in the sexual expression of C. pepo was not so evident. The application of brassinazole, an inhibitor of brassinosteroid biosynthesis slightly changes the production of ethylene in the three analysed genotypes, but those changes have little effect on their sexual phenotypes, and they do not alter the development of the unisexual flowers.     

Two androecious mutations reveal the crucial role of ethylene receptors in the initiation of female flower development in Cucurbita pepo

Ethylene is the key regulator of sex determination in monoecious species of the family Cucurbitaceae. This hormone determines which individual floral meristems develop as female or male flowers and the female flowering transition. The sex determination genes discovered so far code for ethylene biosynthesis enzymes, but little is known about the importance of ethylene signaling components. In this paper we characterize two novel ethylene‐insensitive mutations (etr1a‐1 and etr1b) which block the female flowering transition of Cucurbita pepo; this makes plants produce male flowers indefinitely (androecy). Two missense mutations in the ethylene‐binding domain of the ethylene receptors CpETR1A or CpETR1B were identified as the causal mutations of these phenotypes by using whole‐genome resequencing. The distinctive phenotypes of single and double mutants for four etr mutations have demonstrated that the final level of ethylene insensitivity depends upon the strength and dosage of mutant alleles for at least three cooperating ETR genes, and that the level of ethylene insensitivity determines the final sex phenotype of the plant. The sex phenotype ranges from monoecy in ethylene‐sensitive wild‐type plants to androecy in the strongest ethylene‐insensitive ones, via andromonoecy in partially ethylene‐insensitive plants. The induction of female flowering transition was found to be associated with upregulation of CpACS11, CpACO2 and CpACS27, three ethylene biosynthesis genes required for female flower development. A model is proposed herein, integrating both ethylene biosynthesis and receptor genes into the genetic network which regulates sex determination in C. pepo.     

A Major Gene Conferring Reduced Ethylene Sensitivity and Maleness in Cucurbita pepo

External treatment with ethylene had indicated earlier that this hormone is the main factor controlling sex determination in Cucurbita pepo. Up to now, however, there was no genetic evidence that supported the relationship between ethylene production, or perception, and sexual expression in this species. Here we demonstrate that the extreme male phenotype of the Vegetable Spaghetti (Veg) inbred line of C. pepo subspecies pepo is determined by a major gene that confers reduced ethylene sensitivity in plants. The production of female flowers in the Veg line is very delayed and reduced with respect to the contrasting Bolognese (Bog) line, ranging between 5 and 35% of female flowers per plant. This enhanced maleness trait segregates as a single gene in the F₂ and backcross (BC) generations, and co-segregates with a weak ethylene-insensitive phenotype in the F₂ population, suggesting that the gene responsible for the Veg phenotype could be the result of a mutation in a receptor or response gene for ethylene. Although the etiolated seedlings of the Veg line, and the most androecious plants in the F₂ generation, produce more ethylene than those of the contrasting line, they are less sensitive to this hormone, as indicated by a weaker triple response and a delayed abscission of ethylene-treated male flowers. Given that the sexual phenotype of F₂ plants is correlated with ethylene sensitivity, with the more sensitive plants producing the higher number of female flowers, our results demonstrate that the ethylene response is directly involved in the control of sex determination in C. pepo. It regulates the induction of female flower production, and therefore the extension of the initial phase of development in which the plant produces only male flowers, as well as the number of female flowers per plant.     

Hormonal control of floral abscission in zucchini squash (<Emphasis Type="Italic">Cucurbita pepo</Emphasis>)

In the zucchini squash, Cucurbita pepo, a well coordinated abscission of the female flower during fruit set is essential to obtain a fruit of commercial value. In Spain zucchini is mainly produced in greenhouses in Almería, where high temperatures during the spring-summer period provoke a cultivar-dependent defect in fruits known as the “sticky flower” syndrome. This disorder is characterised by an arrest in growth and maturation of floral organs, and a lack of female floral abscission, thus diminishing fruit shelf-life, commercial quality and value. The aim of the present work was to improve knowledge of the abscission process in C. pepo to better understand the fundamental causes of this disorder. The anatomical analysis of abscission shows a well defined male floral abscission zone (AZ), few hours after anthesis, which differs from the female zone which is not differentiated from the adjacent tissue until the abscission process has begun, and which occurs as a consequence of AZ cell enlargement and the dissolution of their cell walls. To evaluate the role of ethylene and auxins in the regulation of floral abscission in zucchini we performed several treatments, with: ethylene, added as 0.25% ethrel solution; AVG, the inhibitor of ethylene synthesis, at 100 μM; indol-3-acetic acid, 100 μM; and TIBA, the inhibitor of auxin polar transport, at 10 mM. These treatments show that ethylene is an accelerator of zucchini floral abscission, and also promotes abscission in isolated AZs of sticky flowers. On the other hand, IAA delays abscission of the female flowers, whilst the inhibitor of auxin polar transport promotes it. The activity of the cell wall hydrolytic enzymes, polygalacturonase and cellulase, sharply increased just before the shedding of zucchini floral organs (72 h after anthesis). Moreover, both enzyme activities were induced by ethylene, which partly explains the ethylene promoting effect.     

Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

To understand the regulatory mechanisms governing unisexual flower development in cucumber, we conducted a systematic morphogenetic analysis of male and female flower development, examined the dynamic changes in expression of the C-class floral organ identity gene CUM1, and assessed the extent of DNA damage in inappropriate carpels of male flowers. Accordingly, based on the occurrence of distinct morphological events, we divided the floral development into 12 stages ranging from floral meristem initiation to anthesis. As a result of our investigation we found that the arrest of stamen development in female flowers, which occurs just after the differentiation between the anther and filament, is mainly restricted to the primordial anther, and that it is coincident with down-regulation of CUM1 gene expression. In contrast, the arrest of carpel development in the male flowers occurs prior to the differentiation between the stigma and ovary, given that no indication of ovary differentiation was observed even though CUM1 gene expression remained detectable throughout the development of the stigma-like structures. Although the male and female reproductive organs have distinctive characteristics in terms of organ differentiation, there are two common features regarding organ arrest. The first is that the arrest of the inappropriate organ does not affect the entirety of the organ uniformly but occurs only in portions of the organs. The second feature is that all the arrested portions in both reproductive organs are spore-bearing parts.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy - TUNEL TdT-mediated dUTP nick-end labeling     

Involvement of Ethylene Biosynthesis and Signalling in the Transition from Male to Female Flowering in the Monoecious Cucurbita pepo

It is well established that ethylene is the main hormonal regulator of sexual expression in the Cucurbitaceae family, controlling not only the sexual fate of individual floral buds, but also the female flower transition, that is, the time at which the first female flower appears and therefore the number of female flowers per plant. Although sex determination of individual flower buds is known to be controlled by specific ethylene biosynthesis ACS genes in melon and cucumber, the role of ethylene genes in the control of the transition to female flowering is still unknown. We have identified two contrasting monoecious inbred lines of Cucurbita pepo, Bolognese (Bog) and Vegetable spaghetti (Veg), which differ in female flower transition but not in flower development. In Bog, which is very sensitive to ethylene, the transition to female flowering is very early, whereas in Veg, which is much less sensitive to ethylene, the transition occurs much later. In this article we compare the production of ethylene and the expression profiles of seven genes involved in the biosynthesis, perception, and signalling of ethylene in the two contrasting lines. Bog, with earlier female flower transition, showed higher ethylene production and CpACO1 expression in the apex at an earlier stage of plant development, when Bog is already producing female flowers, but Veg has not transitioned to female flowering yet. Moreover, the expression of the ethylene receptor and CTR-like genes in the apex of Veg and Bog plants indicates that these genes negatively regulate female flower transition during the earlier stages of plant development. The earlier transition to female flowering in Bog is not only associated with a higher production of ethylene in the apex but also with a premature decline of ethylene negative regulators (receptors and CTR-like) in the apex of the plant. These results provide the basis for a model that explains the regulation of female flowering transition in monoecious cucurbits.     

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。     

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     

Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.)

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

蝴蝶兰花发育的分子生物学研究进展

蝴蝶兰花非常独特且高度进化,如萼片瓣化、瓣片特化为唇瓣、雌雄蕊合生成合蕊柱及子房发育须由授粉启动等,是单子叶植物花发育研究的理想材料。近年来蝴蝶兰花发育分子生物学取得了重要进展。该文就近年来国内外有关蝴蝶兰开花转换及花器官发育相关基因研究以及B类基因与兰花花被的进化发育关系方面的研究进展进行综述。研究表明:MADS基因在蝴蝶兰开花转换及花器官发育过程中起重要作用,推测其中的DEF(DE-FICIENS)-like基因早期经过2轮复制,形成了4类不同的DEF-like基因,进而决定兰花花被属性。蝴蝶兰花发育分子生物学的深入研究,将极大地利于通过基因工程手段提高蝴蝶兰花品质如花色改良及花期调控等,推动分子育种进程。     

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.     

Gymnosperm Orthologues of Class B Floral Homeotic Genes and Their Impact on Understanding Flower Origin

Class B floral homeotic genes play a key role in specifying the identity of male reproductive organs (stamens) and petals during the development of flowers. Recently, close relatives (orthologues) of these genes have been found in diverse gymnosperms, the sister group of the flowering plants (angiosperms). The fact that such genes have not been found so far, despite considerable efforts, in mosses, ferns or algae, has been taken as evidence to suggest that B genes originated 300–400 million years ago in a lineage that led to extant seed plants. Gymnosperms do not develop petals, and their male reproductive organs deviate considerably from angiosperm stamens. So what is the function of gymnosperm B genes? Recent experiments revealed that B genes from diverse extant gymnosperms are exclusively expressed in male reproductive organs (microsporophylls). At least for some of these genes it has been shown that they can partially substitute for the Arabidopsis B genes AP3 and PI in ectopic expression experiments, or even partially substitute these genes in different class B floral organ identity gene mutants. This functional complementation, however, is restricted to male organ development. These findings strongly suggest that gymnosperm and angiosperm B genes have highly related interaction partners and equivalent functions in the male organs of their different host species. It seems likely that in extant gymnosperms B genes have a function in specifying male reproductive organs. This function was probably established already in the most recent common ancestor of extant gymnosperms and angiosperms (seed plants) 300 million years ago and thus represents the ancestral function of seed plant B genes, from which other functions (e.g., in specifying petal identity) might have been derived. This suggests that the B gene function is part of an ancestral sex determination system in which B gene expression specifies male reproductive organ development, while the absence of B gene expression leads to the formation of female reproductive organs. Such a simple switch mechanism suggests that B genes might have played a central role during the origin of flowers. In the out-of-male and out-of-female hypotheses changes in B gene expression led to the origin of hermaphroditic flower precursors out of male or female gymnosperm reproductive cones, respectively. We compare these hypotheses with other recent molecular hypotheses on the origin of flowers, in which C/D and FLORICAULA/LEAFY-like genes is given a more prominent role, and we suggest how these hypotheses might be tested in the future.     

Stamen development in <Emphasis Type="Italic">Arabidopsis</Emphasis> is arrested by organ-specific overexpression of a cucumber ethylene synthesis gene <Emphasis Type="Italic">CsACO2</Emphasis>

Cucumber (Cucumis sativus L.) has served as a model to understand hormone regulation in unisexual flower development since the 1950s and the role of ethylene in promoting female flower development has been well documented. Recent studies cloned the F-locus in gynoecious lines as an additional copy of the ACC synthase (ACS) gene, which further confirmed the role of ethylene in the promotion of female cucumber flowers. However, no direct evidence was generated to demonstrate that increases in endogenous ethylene production could induce female flowers by arresting stamen development. To clarify the relationship between ethylene production and stamen development, we overexpressed the ethylene synthesis cucumber gene CsACO2 to generate transgenic Arabidopsis, driven by the organ-specific promoter P _AP3 . We found that organ-specific overexpression of CsACO2 significantly affected stamen but not carpel development, similar to that in the female flowers of cucumber. Our results suggested that increases in ethylene production directly disturb stamen development. Additionally, our study revealed that among all floral organs, stamens respond most sensitively to exogenous ethylene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of modified endogenous ethylene production on sex expression, bisexual flower development and fruit production in melon (Cucumis melo L.)

Members of the Cucurbitaceae family display a range of sexual phenotypes including various combinations of male, female, or bisexual flowers. Ethylene appears to be a key hormone regulating the sex determination process. Application of ethylene, or inhibition of ethylene action, increases or decreases the number of pistil-bearing buds, respectively. Elevated levels of ethylene production and expression of genes for ethylene biosynthesis, have been correlated with pistillate flower production. In this study, we sought to determine the effect of modified endogenous ethylene production on sex expression by constitutively expressing ACS (1-aminocyclopropane-1-carboxylate synthase), the first committed enzyme for ethylene biosynthesis, in transgenic melons (Cucumis melo L.). Most melon genotypes are andromonoecious, where an initial phase of male flowers is followed by a mixture of bisexual and male flowers. ACS melon plants showed increased ethylene production by leaves and flower buds, and increased femaleness as measured by earlier and increased number of bisexual buds. ACS melons also had earlier and increased number of bisexual buds that matured to anthesis, suggesting that ethylene is important not only for sex determination, but also for development of the bisexual bud to maturity. Field studies showed that ACS melons had earlier mature bisexual flowers, earlier fruit set, and increased number of fruit set on closely spaced nodes on the main stem. These results provide a direct demonstration of the importance of endogenous ethylene production for female reproductive processes in melon.     

Changes in polyamine pattern mediates sex differentiation and unisexual flower development in monoecious cucumber (Cucumis sativus L.)

Changes in the levels of polyamines are associated with fundamental physiological processes such as embryogenesis, induction of flowering, fruit development and ripening, senescence, and responses to environmental stresses, but the role of polyamines in sex differentiation and unisexual flower development has not been deeply studied. To extend the knowledge on the regulatory mechanisms of flowering in monoecious plant (producing unisexual flowers), we investigated the morphogenesis and free polyamine levels in Cucumis sativus during sex differentiation and unisexual flower development in vitro using histocytological and biochemical methods. As shown in our study, floral development in vitro was undisturbed and flowers of both sexes were produced. Sex differentiation relied on preventing the development of generative organs of the opposite sex, as we observed carpel repression in male flowers and stamen repression in female flowers. Pollen viability was negatively correlated with female flower development on the same node. Biochemical analysis revealed increased accumulation of aliphatic amines (tri, tetra‐amines) in generative (flower buds and flowers) compare to vegetative (axillary buds and leaves) organs. Undifferentiated floral buds contained elevated levels of agmatine, cadaverine, spermidine and spermine. Sex differentiation was associated with significantly decreased levels of agmatine and cadaverine. Our results showed that female flowers contained higher levels of total polyamine than male flowers. The increased level of cadaverine was associated with macrogametogenesis and female flower maturation. Putrescine was important for male flower development. Such results support the hypothesis that aliphatic amines are involved in unisexual flower development.     

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.