Genomic organization of the AODEF gene in Asparagus officinalis L

The perianths of Liliaceae plants, such as lily and tulip, have two whorls of almost identical petaloid organs, which are called tepals. According to the modified ABC model proposed in tulip, the class B genes are expressed in whorl 1 as well as whorls 2 and 3, so that the organs of whorls 1 and 2 have the same petaloid structure. The floral structure of asparagus (Asparagus officinalis L.) is similar to that of Liliaceae plants, however, the expression of B-class genes (AODEF, AOGLOA, AOGLOB) was not found in whorl 1, but was confined to whorls 2 and 3. This result does not support the modified ABC model in asparagus. In order to gain a better understanding of asparagus flower development, we have characterized a genomic clone of the AODEF gene. We compared the genomic organization and promoter sequence of AODEF with three well-studied DEF-like genes, DEFICIENS (Antirrhinum), APETALA3 (Arabidopsis), and OSMADS16 (rice). Exon-intron structures of these genes are well-conserved except for the large fifth intron in the AODEF gene and the OSMADS16 gene. Putative cis-elements including CArG-boxes were found in the promoter region and forty-two microsatellites were found in the AODEF genomic sequence.     

Two GLOBOSA-like genes are expressed in second and third whorls of homochlamydeous flowers in Asparagus officinalis L

Garden asparagus (Asparagus officinalis L.) has homochlamydeous flowers. Like Liliaceae plants such as lily and tulip, the perianths of asparagus have two whorls of almost identical petaloid organs, called tepals. Floral structures of these homochlamydeous flowers could be explained by a modified ABC model, in which the expression of the class B genes has expanded to whorl 1, so that the organs of whorls 1 and 2 have the same petaloid structure. In this study, we isolated and characterized two GLOBOSA-like genes (AOGLOA and AOGLOB), one of class B gene, from asparagus. Southern blot showed that AOGLOA and AOGLOB genes are single copy genes. Northern blot analysis indicated that these genes were specifically expressed in male and female flowers. In situ hybridization showed that the expression of AOGLOA and AOGLOB genes is confined to whorls 2 and 3 (inner tepal and stamen) and not detected in whorl 1 (outer tepal). The other asparagus class B gene, AODEF, was also not expressed in outer tepal [Park et al. (2003) Plant Mol Biol. 51: 867]. These results indicate that the class B genes are not involved in the outer tepal development in asparagus, not supporting the modified ABC model in asparagus.     

Floral organogenesis in Tetracentron sinense (Trochodendraceae) and its systematic significance

In Tetracentron sinense of the basal eudicot family Trochodendraceae, the flower primordium, together with the much retarded floral subtending bract primordium appear to form a common primordium. The four tepals and the four stamens are initiated in four distinct alternating pairs, the first tepal pair is in transverse position. The four carpels arise in a whorl and alternate with the stamens. This developmental pattern supports the interpretation of the flower as dimerous in the perianth and androecium, but tetramerous in the gynoecium. There is a relatively long temporal gap between the initiation of the stamens and the carpels. The carpel primordia are then squeezed into the narrow gaps between the four stamens. In contrast to Trochodendron, the residual floral apex after carpel formation is inconspicuous. In their distinct developmental dimery including four tepals and four stamens, flowers of Tetracentron are reminiscent of other, related basal eudicots, such as Buxaceae and Proteaceae.     

The modified ABC model explains the development of the petaloid perianth of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> ssp. <Emphasis Type="Italic">orientalis</Emphasis> (Agapanthaceae) flowers

The class B genes, which belong to the MADS-box gene family, play important roles in regulating the development of petals and stamens in flowering plants. To understand the molecular mechanisms of floral development in Agapanthus praecox ssp. orientalis (Agapanthaceae), we isolated and characterized the homologs of the Antirrhinum majus genes GLOBOSA and DEFICIENS in this plant. These were designated as ApGLO and ApDEF, respectively. ApGLO and ApDEF contain open reading frames that encode deduced protein with 210 and 214 amino acid residues, respectively. Phylogenetic analysis indicated that ApGLO and ApDEF belong to the monocot class B gene family. In situ hybridization experiments revealed that hybridization signals of ApGLO and ApDEF were observed in whorl 1 as well as in whorls 2 and 3. Moreover, the flowers of transgenic Arabidopsis plants that ectopically expressed ApGLO formed petal-like organs in whorl 1. These observations indicate that the flower developmental mechanism of Agapanthus follows the modified ABC model.     

Within and between Whorls: Comparative Transcriptional Profiling of Aquilegia and Arabidopsis

Background

The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels.

Methodology/Principal Findings

We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource).

Conclusions/Significance

Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.     

B-class MADS-box genes in trioecious papaya: two paleoAP3 paralogs, CpTM6-1 and CpTM6-2, and a PI ortholog CpPI

In the ABC model of flower development, B function organ-identity genes act in the second and third whorls of the flower to control petal and stamen identity. The trioecious papaya has male, female, and hermaphrodite flowers and is an ideal system for testing the B-class gene expression patterns in trioecious plants. We cloned papaya B-class genes, CpTM6-1, CpTM6-2, and CpPI, using MADS box gene specific degenerate primers followed by cDNA library screening and sequencing of positive clones. While phylogenetic analyses show that CpPI is the ortholog of the Arabidopsis gene PI, the CpTM6-1 and CpTM6-2 loci are representatives of the paralogous TM6 lineage that contain paleoAP3 motifs unlike the euAP3 gene observed in Arabidopsis. These two paralogs appeared to have originated from a tandem duplication occurred approximately 13.4 million year ago (mya) (bootstrap range 13.36 ± 2.42). In-situ hybridization and RT-PCR showed that the papaya B-class genes were highly expressed in young flowers across all floral organ primordia. As the flower organs developed, all three B-class genes were highly expressed in petals of all three-sex types and in stamens of hermaphrodite and male flowers. CpTM6-1 expressed at low levels in sepals and carpels, whereas CpTM6-2 expressed at a low level in sepals and at a high level in leaves. Our results showed that B-class gene homologs could function as predicted by the ABC model in trioecous flowers but differential expressions of CpTM6-1, and CpTM6-2, and CpPI suggested the diversification of their functions after the duplication events. Christine M. Ackerman, Qingyi Yu contributed equally to this work.     

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.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.     

Conservation of the E-function for Floral Organ Identity in Rice Revealed by the Analysis of Tissue Culture-induced Loss-of-Function Mutants of the <Emphasis Type="Italic">OsMADS1</Emphasis> Gene

Rapid progress in studies on flower development has resulted in refining the classical ‘ABC model’ into a new ‘ABCDE model’ to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicotyledonous (dicot) plants has been revealed. However, its conservation in monocotyledonous (monocot) plants remains largely unknown. Here, we show the conservation of E-function in rice (Oryza sativaL.) by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123(SEPALLATA1/2/3) and the petunia FBP2(Floral Binding Protein 2), suggesting that OsMADS1play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice, a monocot model plant.     

The homeotic Macho mutant of Antirrhinum majus reverts to wild-type or mutates to the homeotic plena phenotype

Plants of Antirrhinum majus carrying the semidominant Macho alleles of the plena gene display carpelloid sepals and staminoid petals, but the two inner flower whorls of stamens and carpels are normal and produce fertile gametes. In the recessive plena mutant, in contrast, the two outer whorls are normal whereas the stamens are largely or entirely petaloid and the carpels sepaloid, thus producing weakly male-fertile or fully sterile lines. Two new plena and two new Macho alleles have been induced in transposon tagging experiments. Genetic and molecular analysis revealed that the two contrasting mutant phenotypes are caused by mutations in one and the same gene: Several wild-type plants appeared among 27 000 F1 plants of a cross between Macho female plants and wild-type males bearing the active transposons Taml and Tam3. One of these plants segregated plena mutants, three showed reversions to wild-type and another two segregated Macho plants, possibly representing somatic reversions. Additional evidence was provided by an allelism test of Macho × plena. Molecular analysis has independently corroborated the genetical results. Moreover, the double mutant Macho/deficiens shows only carpels and plena/deficiens only sepals, which is in accord with combinatorial models for homeotic flower formation presented recently.     

Developmental morphology of the flower of <Emphasis Type="Italic">Dracontium polyphyllum</Emphasis> in the context of the phylogeny of the Araceae

The inflorescence of Dracontium polyphyllum consists of 150 – 300 flowers arranged in recognisable spirals. The flower has 5 – 6 (90% of observed specimens), or 7 broad tepals enclosing 9 – 12 stamens (occasionally 7) inserted in two whorls. The gynoecium is trilocular (90% of observed specimens) or tetralocular. The tetralocular gynoecia are found at random among the trilocular gynoecia. Each locule encloses an ovule inserted in an axile position, in the median portion of the ovary. Each carpel has its own stylar canal. However, in the upper portion of the style, there is only one common stylar canal. Floral organs are initiated in an acropetal direction in the following sequence: tepals, stamens, and carpels. During later stages of development, the tepals progressively cover the other floral organs. The first floral primordia are initiated on the upper portion of the inflorescence. During early stages of development, the floral primordia have a circular shape. The tepals are initiated nearly simultaneously. During later stages of development, the first whorl of stamens develops in alternation with the tepals and is followed by a second whorl of stamens. The trilocular or tetralocular nature of the ovary is clearly visible during early stages of development of the gynoecium. Recent molecular studies show that Anaphyllopsis A. Hay and Dracontium L. are closely related. However, although pentamerous flowers have been observed in Anaphyllopsis, the developmental morphology of the flower of Dracontium is different from that of Anaphyllopsis.     

Inflorescence and floral ontogeny in Crocus sativus L. (Iridaceae)

Inflorescence and floral ontogeny of the perennial, herbaceous crop Crocus sativus L. were studied using epi-illumination light microscopy. After production of leaves with helical arrangement a determinate inflorescence forms which becomes completely transformed into a single terminal flower. In some cases, bifurcation of the inflorescence meristem yields two or three floral meristems. The order of floral organs initiation is outer tepals – stamens – inner tepals – carpels. Stamens and outer tepals are produced from the lateral bifurcation of three common stamen-tepal primordia. Within each whorl, organs start developing unidirectionally from the adaxial side, except for the stamens which begin to grow from the abaxial side. Specialized features during organ development include interprimordial growth between tepals forming a perianth tube, fusion at the base of stamen filaments, and formation of an inferior ovary with unfused styles.     

The corona of the daffodil Narcissus bulbocodium shares stamen‐like identity and is distinct from the orthodox floral whorls

The structural homology of the daffodil corona has remained a source of debate throughout the history of botany. Over the years it has been separately referred to as a modified petal stipule, stamen and tepal. Here we provide insights from anatomy and molecular studies to clarify the early developmental stages and position of corona initiation in Narcissus bulbocodium. We demonstrate that the corona initiates as six separate anlagen from hypanthial tissue between the stamens and perianth. Scanning electron microscope images and serial sections demonstrate that corona initiation occurs late in development, after the other floral whorls are fully developed. To define more precisely the identity of the floral structures, daffodil orthologues of the ABC floral organ identity genes were isolated and expression patterns were examined in perianth, stamens, carpel, hypanthial tube and corona tissue. Coupled with in situ hybridisation experiments, these analyses showed that the expression pattern of the C‐class gene NbAGAMOUS in the corona is more similar to that of the stamens than that of the tepals. In combination, our results demonstrate that the corona of the daffodil N. bulbocodium exhibits stamen‐like identity, develops independently from the orthodox floral whorls and is best interpreted as a late elaboration of the region between the petals and stamens associated with epigyny and the hypanthium.     

A B Functional Gene Cloned from Lily Encodes an Ortholog of <Emphasis Type="BoldItalic">Arabidopsis PISTILLATA</Emphasis> (<Emphasis Type="BoldItalic">PI</Emphasis>)

The classical ABC model proposed for flower development in Arabidopsis and Antirrhinum appropriately sheds light on the biological process of flower development and differentiation and serves in manipulating the floral structure of other important ornamental plants. In this study, LLGLO1, a B functional gene from Lilium longiflorum was isolated and characterized. RT-PCR analysis elucidated that temporal and spatial expression pattern of LLGLO1. This putative gene was strongly expressed in 1, 2, and 3 whorl organs, i.e., outer whorl tepals, inner whorl tepals, and stamens. Genetic effect of LLGLO1 was assayed by ectopic expression in model plant Arabidopsis. Transformed plants showed homeotic transformation of sepals into petaloid sepals in the first whorl, which is similar to the transgenic plants of 35S::PI. So LLGLO1 was one member of GLO/PI sub-family gene to function in flower development.     

Development of flowers and inflorescences of Circaeaster (Circaeasteraceae, Ranunculales)

The flower organs of Circaeaster are spiral in origin. The primordia of the tepals, stamens and carpels are almost the same in shape and size in early development. Carpel conduplication takes place only in the middle part of the carpel. The basal part of the carpel differentiates into a short stalk and the upper part into the style and the stigma. Flower development is similar to that of Kingdonia. Floral development of Circaeaster is compared with that of other Ranunculales.     

Co-suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem

The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.     

基于扫描电镜技术的天葵属(毛茛科)花器官发生研究

毛茛科天葵属为东亚特有类群,但其花器官的发生过程仍不清晰。该研究利用扫描电子显微镜观察了天葵［S. adoxoides (DC.) Makino］花器官的发生过程,以揭示毛茛科花形态的多样性和演化规律,为进一步探讨天葵属与近缘类群的亲缘关系提供发育形态学证据。结果表明：(1)天葵萼片、花瓣和雄蕊均为螺旋状发生,轮状排列;不育雄蕊的数目和位置不定,心皮轮状发生。(2)天葵萼片原基为宽阔的新月形,其他花器官为窄的半球形。(3)天葵花发育后期,花瓣有延迟发育现象,花瓣原基基部发育为浅囊状,心皮原基马蹄形对折,胚珠倒生、双珠被、具胎座附属物。(4)天葵属与耧斗菜属、尾囊草属的花发育性状存在相似性,支持分子系统学证据的三者近缘的观点;天葵属的花性状的特殊表现为：花直径较小,雄蕊、不育雄蕊和心皮数目较少,花器官没有形成明显的直列线,内珠被较长等。     

THE DEVELOPMENTAL BASIS OF TRISTYLY IN EICHHORNIA PANICULATA (PONTEDERIACEAE)

Eichhornia paniculata is a tristylous, self-compatible, emergent aquatic. A given plant produces flowers with either long, mid or short styles and two levels of stamens equal in length to the styles not found in that flower. Flowers of each morph have two whorls of three tepals, six stamens and three fused carpels. The six stamens differentiate into two sets of three stamens each. A relatively short set, having either short- or mid-level stamens, occurs on the upper side of the flower, while a relatively long set, having either mid- or long-level stamens, occurs on the lower side. Stamen level depends on differences among stamens in filament length and position of insertion on the floral tube. Floral parts arise in whorls of three, but the two stamen whorls do not form the two sets of stamens found in each mature flower. Instead, stamens from both whorls make up a given set. Floral differences among morphs are not present at flower origin or floral organ initiation. Morphological differences arise first among stamen sets. The two sets within a flower differ prior to meiosis in the size, number, and timing of comparable developmental events in the sporogenous cells. After these initial differences arise, anther size diverges. In later developmental stages differences in filament and floral tube length, cell size, and cell number, as well as differences in the length, cell size, and cell number of styles, develop among morphs. This sequence of developmental events suggests that the genes controlling development in different morphs do not control flower and floral organ initiation but are first morphologically visible in sporogenous cell differentiation.