Ectopic expression of SUPERMAN suppresses development of petals and stamens

The floral regulatory gene SUPERMAN (SUP) encodes a C2H2 type zinc finger protein that is required for maintaining boundaries between floral organs in Arabidopsis. It has been proposed that the main function of SUP is to balance cell proliferation in the third and fourth whorl of developing flowers, thereby maintaining the boundaries between the two whorls. To gain further insight into the function of SUP, we have ectopically expressed SUP using the promoter of APETALA1 (AP1), a gene that is initially expressed throughout floral meristems and later becomes restricted to the first and second whorls. Flowers of AP1::SUP plants have fewer floral organs, consistent with an effect of SUP on cell proliferation. In addition, the AP1::SUP transgene caused the conversion of petals to sepals and suppressed the development of stamens. The expression of the B function homeotic gene APETALA3 (AP3) and its regulator UNUSUAL FLORAL ORGANS (UFO) were delayed and reduced in AP1::SUP flowers. However, SUP does not act merely through UFO, as constitutive expression of UFO did not rescue the defects in petal and stamen development in AP1::SUP flowers. Together, these results suggest that SUP has both indirect and direct effects on the expression of B function homeotic genes.     

Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function

The phytohormone gibberellin (GA) regulates the development and fertility of Arabidopsis flowers. The mature flowers of GA-deficient mutant plants typically exhibit reduced elongation growth of petals and stamens. In addition, GA-deficiency blocks anther development, resulting in male sterility. Previous analyses have shown that GA promotes the elongation of plant organs by opposing the function of the DELLA proteins, a family of nuclear growth repressors. However, it was not clear that the DELLA proteins are involved in the GA-regulation of stamen and anther development. We show that GA regulates cell elongation rather than cell division during Arabidopsis stamen filament elongation. In addition, GA regulates the cellular developmental pathway of anthers leading from microspore to mature pollen grain. Genetic analysis shows that the Arabidopsis DELLA proteins RGA and RGL2 jointly repress petal, stamen and anther development in GA-deficient plants, and that this function is enhanced by RGL1 activity. GA thus promotes Arabidopsis petal, stamen and anther development by opposing the function of the DELLA proteins RGA, RGL1 and RGL2.     

Restoration of stamen development and production of functional pollen in an alloplasmic CMS tobacco line by ectopic expression of the Arabidopsis thaliana SUPERMAN gene

The alloplasmic male-sterile tobacco line Nta(rep)S, combining the nucleus of Nicotiana tabacum with the cytoplasm of Nicotiana repanda, exhibits cadastral-type anomalies due to a fusion of several stamens with the pistil. These anomalies share similarities with Arabidopsis superman mutants. SUPERMAN (SUP) is a cadastral gene controlling the boundary between whorls 3 (androecium) and 4 (gynoecium). Thus we hypothesized that the expression of the tobacco SUP orthologue might be impaired in the alloplasmic Nta(rep)S line, and that the deficiency could be complemented by the Arabidopsis SUP gene. Here we show that the ectopic expression of SUP in the alloplasmic male-sterile tobacco line Nta(rep)S significantly increases the frequency of flowers possessing free stamens, inducing the recovery of a proper structure for whorls 3 and 4. Furthermore, flowers of transgenic plants show a significant improvement of the morphology of stamens, and more particularly of the anthers, which are able to produce few but functional pollen. The data show that ectopic expression of Arabidopsis SUP reactivates the regulatory cascade of anther development. The plausible causes of the developmental defects of anthers in the alloplasmic male-sterile tobacco line are discussed in relation to the model of regulation of the Arabidopsis SUP gene.     

The Arabidopsis SUPERMAN Gene Mediates Asymmetric Growth of the Outer Integument of Ovules

Arabidopsis superman (sup, also referred to as floral mutant10) mutants have previously been shown to have flowers with supernumerary stamens and reduced carpels as a result of ectopic expression of the floral homeotic gene APETALA3 (AP3). Here, we report that sup mutations also cause specific alterations in ovule development. Growth of the outer integument of wild-type ovules occurs almost exclusively on the abaxial side of the ovule, resulting in a bilaterally symmetrical hoodlike structure. In contrast, the outer integument of sup mutant ovules grows equally on all sides of the ovule, resulting in a nearly radially symmetrical tubular shape. Thus, one role of SUP is to suppress growth of the outer integument on the adaxial side of the ovule. Genetic analyses showed that the effects of sup mutations on ovule development are independent of the presence or absence of AP3 activity. Thus, SUP acts through different mechanisms in its early role in ensuring proper determination of carpel identity and in its later role in asymmetric suppression of outer integument growth.     

The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development

In both Antirrhinum (Antirrhinum majus) and Arabidopsis (Arabidopsis thaliana), the floral B-function, which specifies petal and stamen development, is embedded in a heterodimer consisting of one DEFICIENS (DEF)/APETALA3 (AP3)-like and one GLOBOSA (GLO)/PISTILLATA (PI)-like MADS box protein. Here, we demonstrate that gene duplications in both the DEF/AP3 and GLO/PI lineages in Petunia hybrida (petunia) have led to a functional diversification of their respective members, which is reflected by partner specificity and whorl-specific functions among these proteins. Previously, it has been shown that mutations in PhDEF (formerly known as GREEN PETALS) only affect petal development. We have isolated insertion alleles for PhGLO1 (FLORAL BINDING PROTEIN1) and PhGLO2 (PETUNIA MADS BOX GENE2) and demonstrate unique and redundant properties of PhDEF, PhGLO1, and PhGLO2. Besides a full homeotic conversion of petals to sepals and of stamens to carpels as observed in phglo1 phglo2 and phdef phglo2 flowers, we found that gene dosage effects for several mutant combinations cause qualitative and quantitative changes in whorl 2 and 3 meristem fate, and we show that the PHDEF/PHGLO1 heterodimer controls the fusion of the stamen filaments with the petal tube. Nevertheless, when the activity of PhDEF, PhGLO1, and PhGLO2 are considered jointly, they basically appear to function as DEF/GLO does in Antirrhinum and to a lesser extent as AP3/PI in Arabidopsis. By contrast, our data suggest that the function of the fourth B-class MADS box member, the paleoAP3-type PETUNIA HYBRIDA TM6 (PhTM6) gene, differs significantly from the known euAP3-type DEF/AP3-like proteins; PhTM6 is mainly expressed in the developing stamens and ovary of wild-type flowers, whereas its expression level is upregulated in whorls 1 and 2 of an A-function floral mutant; PhTM6 is most likely not involved in petal development. The latter is consistent with the hypothesis that the evolutionary origin of the higher eudicot petal structure coincided with the appearance of the euAP3-type MADS box genes.     

Cucumber SUPERMAN Has Conserved Function in Stamen and Fruit Development and a Distinct Role in Floral Patterning

The Arabidopsis SUPERMAN (SUP) gene encodes a C2H2 type zinc finger protein that is required for maintaining the boundaries between stamens and carpels, and for regulating development of ovule outer integument. Orthologs of SUP have been characterized in bisexual flowers as well as dioecious species, but it remains elusive in monoecious plants with unisexual flowers on the same individual. Here we isolate the SUP ortholog in Cucumis sativus L (CsSUP), a monoecious vegetable. CsSUP is predominantly expressed in female specific organs: the female flower buds and ovules. Ectopic expression of CsSUP in Arabidopsis can partially complement the fruit development in sup-5 mutant, and its over-expression in wide-type leads to reduced silique length, suppressed stamen development and distorted petal patterning. Our data suggest that CsSUP plays conserved as well as distinct roles during flower and fruit development, and it may function in the boundaries and ovules to balance petal patterning, stamen and ovule development in Arabidopsis.     

Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco

Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over-expressed the SUP gene in petunia and tobacco in a tissue-specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over-expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion.     

Arabidopsis (Brassicaceae) flower development and gynoecium patterning in wild type and Ettin mutants

Screening for mutations that alter flower development in Arabidopsis has led to the identification of two general types of genetic loci: those affecting meristem and organ identity, and those affecting growth and development independent of identity. ettin (ett) mutants belong to the latter class and exhibit pleiotropic phenotypes distinct from previously described Arabidopsis mutants. These phenotypes include increases in sepal and petal number, decreases in stamen number and anther locule number, and gross alteration of tissue patterning in the gynoecium. To determine when and how differences in ett floral meristems originate, flower development was compared between the wild type and ett mutants. ett floral meristems exhibit increases in abaxial sepal and petal primordia number without apparent increases in meristem size. Extra sepal and petal primordia develop into normal organs. In contrast, stamen and carpel primordia exhibit alterations in shape and form, subsequent to premature elongation of the terminal floral meristem. Phenotypes are allele-strength dependent. The stigma develops precociously and style differentiation is basally and abaxially misplaced in ett gynoecia. The data are discussed in the context of a model suggesting that two concentric boundaries specify the apical-basal pattern of gynoecium differentiation.     

LEAFY Interacts with Floral Homeotic Genes to Regulate Arabidopsis Floral Development

In the leafy mutant of Arabidopsis, most of the lateral meristems that are fated to develop as flowers in a wild-type plant develop as inflorescence branches, whereas a few develop as abnormal flowers consisting of whorls of sepals and carpels. We have isolated several new alleles of leafy and constructed a series of double mutants with leafy and other homeotic mutants affecting floral development to determine how these genes interact to specify the developmental fate of lateral meristems. We found that leafy is completely epistatic to pistillata and interacts additively with agamous in early floral whorls, whereas in later whorls leafy is epistatic to agamous. Double mutants with leafy and either apetala1 or apetala2 showed a complete loss of the whorled phyllotaxy, shortened internodes, and suppression of axillary buds typical of flowers. Our results suggest that the products of LEAFY, APETALA1, and APETALA2 together control the differentiation of lateral meristems as flowers rather than as inflorescence branches.     

Trends in evolution of floral ontogeny in Cassia sensu stricto,Senna, and Chamaecrista (Leguminosae: Caesalpinioideae: Cassieae: Cassiinae); a study in convergence

Distinctions in floral ontogeny among three segregate genera (Cassia sensu stricto, Chamaecrista, and Senna) of Cassia L. support their separation. In all species studied, the order of floral organ initiation is: sepals, petals, antesepalous stamens plus carpel, and lastly antepetalous stamens. Sepal initiation is helical in all three genera, which however differ in whether the first sepal is initiated in median abaxial position (Senna), or abaxial and off-median (Cassia javanica), a rare character state among legumes. Order of petal initiation varies: helical in Senna vs. unidirectional in Cassia and Chamaecrista. Both stamen whorls are uniformly unidirectional. Intergeneric ontogenetic differences occur in phyllotaxy, inflorescence architecture, bracteole formation, overlap of initiation among organ whorls (calyx/corolla in Cassia; two stamen whorls in Chamaecrista), eccentric initiation on one side of a flower, anther attachment, anther pore structure, and precocious carpel initiation in Senna. The asymmetric corolla and androecium in Chamaecrista arise by precocious organ initiation on one side (left or right). The poricidal anther character can result from differing developmental pathways: lateral slits vs. sealing of lateral sutures; clasping hairs vs. sutural ridges; terminal pores (one or two) vs. none; and clamp layer formation internally that prevents lateral dehiscence. Genera differ in corolla aestivation patterns and in stigma type. Convergence is shown among the three genera, based on intergeneric dissimilarities in early floral ontogeny (floral position in the inflorescence, bracteole presence, position of the first sepal initiated, order of petal initiation, asymmetric initiation, overlap between whorls, anther morphology, and time of carpel initiation) resulting in similarities at anthesis (showy, mostly yellow salverform flowers, heteromorphic stamens, poricidal anther dehiscence, bee pollination, and chambered stigma).     

Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product.

We characterized the distribution of AGAMOUS (AG) RNA during early flower development in Arabidopsis. Mutations in this homeotic gene cause the transformation of stamens to petals in floral whorl 3 and of carpels to another ag flower in floral whorl 4. We found that AG RNA is present in the stamen and carpel primordia but is undetectable in sepal and petal primordia throughout early wild-type flower development, consistent with the mutant phenotype. We also analyzed the distribution of AG RNA in apetela2 (ap2) mutant flowers. AP2 is a floral homeotic gene that is necessary for the normal development of sepals and petals in floral whorls 1 and 2. In ap2 mutant flowers, AG RNA is present in the organ primordia of all floral whorls. These observations show that the expression patterns of the Arabidopsis floral homeotic genes are in part established by regulatory interactions between these genes.     

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo.     

Arabidopsis and Tobacco superman regulate hormone signalling and mediate cell proliferation and differentiation

Arabidopsis thaliana superman (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation.     

掌叶木的花器官发生及其系统学意义

利用扫描电子显微镜和光学显微镜观察了掌叶木的花器官发生过程。观察结果表明: 花序原基最先发生, 然后形成两个大小不一的花原基; 萼片原基的发生不同步, 螺旋状向心发生; 4-5枚花瓣原基以接近轮状方式近同时发生; 不存在花瓣-雄蕊复合原基; 7-8枚雄蕊原基为近同时发生, 其生长较花瓣原基快; 心皮原基最后发生, 3枚心皮原基为同时发生。花为单性花。在雌花中, 子房膨大而雄蕊退化。在雄花中, 雄蕊正常发育, 子房退化。讨论了掌叶木花器官发生和发育的系统学意义。     

Separable roles of UFO during floral development revealed by conditional restoration of gene function

The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.     

Correlation of microsporangial and megasporangial differentiation along racemes of Phytolacca americana L. (Phytolaccaceae)

Both quantitative and qualitative data were obtained for anthers and ovules throughout the lengths of flowering Phytolacca americana (pokeweed) inflorescences. Comparative data were collected for micro-and megasporangia at seven different locations along the lengths of 20 cm racemes. Each location represented a different developmental stage, from sporangial initiation through full differentiation, along the indeterminate racemes. Reproductive biology of pokeweed anthers, as recognized by inflorescence analysis, was most promoted in development between 4 cm (stage 1) and 5 cm (stage 2) from the terminus of inflorescences. Sporogenous cells first differentiated and stamen cell number quadrupled in stage two. Mitotic divisions, within anther subsurface cells, continued throughout all seven inflorescence stages, while divisions slowed between 6.2 cm (stage 4) and 7 cm (stage 5) from the terminus in anther surface cells. To regulate ovule development maximally, by the treatment of inflorescences, one should manipulate older, more basal flowers (and thus older developmental stages) in excess of 11.6 cm (stage 6) from the terminus. Megasporocytes were initiated and a %fold increase in ovule cell number occurred during stage 6. Similar to anthers, mitosis continued throughout the length of racemes in nucellar cells. Ovule surface cells, however, became mitotically quiescent 11.6 cm (stage 6) from the terminus of racemes.     

基于同质园栽培的21种淫羊藿属植物花部特征的分类学意义

以采自中国多地并同质园栽培于吉首大学种质资源圃的21种淫羊藿为研究材料,通过观测这些物种的花梗长、花直径、内萼片长宽、蜜距长、雌雄蕊长、花药长、花柱长、胚珠数、花粉大小及花粉量等16个花部特征数量性状和花序类型、有无蜜距、花瓣颜色、内萼片颜色、内萼片与花瓣相对长度及花粉颜色6个质量性状,并对其分类学意义进行探讨。结果表明,(1)21种淫羊藿内轮萼片花瓣状,4枚花瓣除无距淫羊藿外均特化成蜜距,但花部形态种间存在显著差异,尤其体现于花直径、内萼片长宽、蜜距长、雌蕊长、胚珠数、花粉量、花粉直径等特征。(2)主成分分析显示花直径、内萼片长、花药长、胚珠数和花粉直径等性状信息负荷量较大,在该属分类中起主要作用。(3)根据花部特征聚类分析,物种间区分良好,21物种可分为4支:第1支花小,花瓣短于内轮萼片,囊状距或兜状距;第2支花较大,花瓣远长于内轮萼片,长距状;第3支花小,花瓣没有特化形成蜜距;第4支花较小,花丝伸长明显,可达10 mm,花蜜距呈钻状。(4)依据所观察到的花部形态特征编写了淫羊藿属21种植物花部特征分种检索表。研究认为,基于同质园栽培下的淫羊藿属物种的花部特征具有分类学意义,可以为该属分类提供一定依据。