Sequence evolution and sex-specific expression patterns of the C class floral identity gene, <Emphasis Type="Italic">SpAGAMOUS</Emphasis>, in dioecious <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L

Development in dioecious cultivated spinach, Spinacia oleracea, is distinguished by the absence of alternative reproductive organ primordia in male and female flowers. Given the highly derived floral developmental program in spinach, we wished to characterize a spinach C class floral identity gene and to determine the patterns of sequence evolution as well as compare the spatial and temporal expression patterns with those of AGAMOUS. The isolated cDNA sequence clusters phylogenetically within the AGAMOUS/FARINELLI C class clade. In comparison with the SLM1 sequence from the related Silene latifolia, amino acid replacements are highly conservative and non-randomly distributed, being predominantly found in hinge regions or on exposed surfaces of helices. The spinach gene (SpAGAMOUS) appears to be exclusively expressed in reproductive tissues and not in vegetative organs. Initial expression of SpAGAMOUS is similar in male and female floral primordia. However, upon initiation of the first whorl organs, SpAGAMOUS becomes restricted to meristemic regions from which the reproductive primordia will develop. This results in an early gender-specific pattern. Thus, the spinach C class gene is differentially expressed prior to reproductive organ development and is, at least, correlated with, if not directly involved in, the sexual dimorphism in spinach.Electronic Supplementary Material Supplementary material is available for this article at     

Functional Conservation of an <Emphasis Type="Italic">AGAMOUS</Emphasis> Orthologous Gene Controlling Reproductive Organ Development in the Gymnosperm Species <Emphasis Type="Italic">Taxus chinensis</Emphasis> var. <Emphasis Type="Italic">mairei</Emphasis>

Arabidopsis AGAMOUS (AG) has roles in specifying reproductive organ (stamens and carpels) identity, floral meristem determinacy, and repression of A-function. To investigate possible roles of AG orthologous genes in gymnosperm species and evolution of C function, we isolated and identified AG orthologous gene TcAG from Taxus chinensis var. mairei (family Taxaceae, order Coniferales), a member of the last divergant lineage from higher Conifer that sisters to Gnetales. Sequence alignment and phylogenetic analysis grouped TcAG into the gymnosperm AG lineage. TcAG was expressed in both developing male and female cones, but there was no expression in juvenile leaves. Ectopic expression of TcAG in an Arabidopsis ag mutant produced flowers with the third whorl petaloid stamen and fourth whorl normal carpel, but failed to convert first whorl sepals into carpeloid organs and second whorl petals into stamenoid organs. A 35S::TcAG transgenic Arabidopsis ag mutant had very early flowering, and produced a misshapen inflorescence with a shortened floral axis. Our results suggest that establishment of the complete C-function occurred gradually during AG lineage evolution even in gymnosperms.     

Functional analysis of an <Emphasis Type="Italic">APETALA1</Emphasis>-like MADS box gene from <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> in regulating floral transition and formation

An Eustoma grandiflorum APETALA1 (EgAP1) gene showing high homology to the SQUA subfamily of MADS-box genes was isolated and characterized. EgAP1, containing a conserved euAP1 motif at the C-terminus, showed high sequence identity to Antirrhinum majus SQUAMOSA in the SQUA subfamily. EgAP1 mRNA was detected in the leaf and expressed significantly higher in young flower buds than in mature flower buds. In flowers, EgAP1 mRNA was strongly detected in sepal, weakly detected in petal and was absent in stamen and carpel. Transgenic Arabidopsis plants ectopically expressing EgAP1 flowered early and produced terminal flowers. In addition, the conversion of petals into stamen-like structures was also observed in 35S::EgAP1 flowers. 35S::EgAP1 was able to complement the ap1 flower defects by restoring the defect for sepal formation and significantly increasing second whorl petal production in Arabidopsis ap1 mutant plants. These results revealed that EgAP1 is the APETALA1 homolog in E. grandiflorum and that the function of EgAP1 is involved in floral induction and flower formation.     

Isolation and characterization of the C-class MADS-box gene from the distylous pseudo-cereal <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

In the model species Arabidopsis thaliana, the floral homeotic C-class gene AGAMOUS (AG) specifies reproductive organ (stamen and carpels) identity and floral meristem determinacy. Gene function analyses in other core eudicots species reveal functional conservation, subfunctionalization and function switch of the C-lineage in this clade. To identify the possible roles of AG-like genes in regulating floral development in distylous species with dimorphic flowers (pin and thrum) and the C function evolution, we isolated and identified an AG ortholog from Fagopyrum esculentum (buckwheat, Family Polygonaceae), an early diverging species of core eudicots preceding the rosids-asterids split. Protein sequence alignment and phylogenetic analysis grouped FaesAG into the euAG lineage. Expression analysis suggested that FaesAG expressed exclusively in developing stamens and gynoecium of pin and thrum flowers. Moreover, FaesAG expression reached a high level in both pin and thrum flowers at the time when the stamens were undergoing rapidly increased in size and microspore mother cells were in meiosis. FaesAG was able to substitute for the endogenous AG gene in specifying stamen and carpel identity and in an Arabidopsis ag-1 mutant. Ectopic expression of FaesAG led to very early flowering, and produced a misshapen inflorescence and abnormal flowers in which sepals had converted into carpels and petals were converted to stamens. Our results confirmed establishment of the complete C-function of the AG orthologous gene preceding the rosids-asterids split, despite the distinct floral traits present in early- and late-diverging lineages of core eudicot angiosperms.     

Floral development of <Emphasis Type="Italic">Gonocaryum</Emphasis> with emphasis on the gynoecium

We investigated the floral development of Gonocaryum, a genus of Cardiopteridaceae that was segregated from Icacinaceae s.l., using scanning electron microscopy to clarify its gynoecial structure and facilitate morphological comparisons of Cardiopteridaceae. The key floral developmental characters include sepal initiation that follows a quincuncial spiral sequence; petals that are valvate with inflexed tips and are postgenitally fused at the base; a petal and stamen initiation sequence that is almost simultaneous; a globular protuberance on top of the connective; a gynoecium that is tricarpellate and pseudomonomerous, with the stigma produced by one abaxial lateral carpel; and two ovules that are unitegmic and anatropous with an obturator on the funicle. The floral developmental characters of Gonocaryum are discussed relative to Cardiopteris, which has been well studied and whose gynoecial vasculature is reinterpreted here, and are briefly compared to other members of Aquifoliales and Icacinaceae s.l. The imbricate sepals, initiated in a quincuncial spiral sequence, and the tricarpellate, pseudomonomerous gynoecium are common characters of Cardiopteridaceae. Unisexual flowers are an autapomorphy of Gonocaryum in Cardiopteridaceae.     

The floral organogenesis of <Emphasis Type="Italic">Koelreuteria bipinnata</Emphasis> and its variety <Emphasis Type="Italic">K. bipinnata</Emphasis> var. <Emphasis Type="Italic">integrifolia</Emphasis> (Sapindaceae): evidence of floral constraints on the evolution of monosymmetry

The floral organogenesis and anatomy of Koelreuteria bipinnata and its variety K. bipinnata var. integrifolia (Sapindaceae) has been investigated to clarify the identity of the two taxa in relation to other species of Koelreuteria, and to understand the shift to monosymmetry in the genus. Although the floral development is highly similar, we found a number of striking differences. Flowers arise in thyrses, with lateral branches forming cincinni of 5–9 flowers. Sepals initiate in a spiral sequence. Five petals arise unidirectionally alternating to the sepals. The last formed petal and one stamen between sepals 3 and 5 are strongly delayed, appearing as a common primordium, while this petal is completely suppressed in var. integrifoliola. Eight stamens initiate sequentially, differ in size and partly precede the development of petals. The gynoecium develops as a triangular primordium on which three carpellary lobes become demarcated simultaneously. Placentation is axile. Septal slits occurring within the style are interpreted as a deep-reaching non-nectariferous extension of the stigma. The massive, oblique disk with crenate apex develops in an extrastaminal position, but is interrupted on the radius of the lost petal. Floral developmental evidence supports variety K. bipinnata var. integrifolia rather than being synonym of K. bipinnata. Floral development is compared with K. paniculata and is discussed in the context of floral evolution of Sapindaceae. Our study demonstrates the importance of developmental shifts on floral evolution. The triangular gynoecium has a strong spatial impact in obliquely reorganizing the symmetry of the flower. It is demonstrated that spatial constraints of calyx and ovary are responsible for the reduction in one of the petals, two stamens and a shift in symmetry of the flower.     

Duplication of <Emphasis Type="Italic">AP1</Emphasis> within the <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. <Emphasis Type="Italic">AP1/FUL</Emphasis> clade is followed by rapid amino acid and regulatory evolution

The AP1/FUL clade of MADS box genes have undergone multiple duplication events among angiosperm species. While initially identified as having floral meristem identity and floral organ identity function in Arabidopsis, the role of AP1 homologs does not appear to be universally conserved even among eudicots. In comparison, the role of FRUITFULL has not been extensively explored in non-model species. We report on the isolation of three AP1/FUL genes from cultivated spinach, Spinacia oleracea L. Two genes, designated SpAPETALA1-1 (SpAP1-1) and SpAPETALA1-2 (SpAP1-2), cluster as paralogous genes within the Caryophyllales AP1 clade. They are highly differentiated in the 3′, carboxyl-end encoding region of the gene following the third amphipathic alpha-helix region, while still retaining some elements of a signature AP1 carboxyl motifs. In situ hybridization studies also demonstrate that the two paralogs have evolved different temporal and spatial expression patterns, and that neither gene is expressed in the developing sepal whorl, suggesting that the AP1 floral organ identity function is not conserved in spinach. The spinach FRUITFULL homolog, SpFRUITFULL (SpFUL), has retained the conserved motif and groups with Caryophyllales FRUITFULL homologs. SpFUL is expressed in leaf as well as in floral tissue, and shows strong expression late in flower development, particularly in the tapetal layer in males, and in the endothecium layer and stigma, in the females. The combined evidence of high rates of non-synonymous substitutions and differential expression patterns supports a scenario in which the AP1 homologs in the spinach AP1/FUL gene family have experienced rapid evolution following duplication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inflorescence and floral development in <Emphasis Type="Italic">Trochodendron aralioides</Emphasis> (Trochodendraceae)

In the early development of Trochodendron aralioides (Trochodendraceae) inflorescences lateral flowers are initiated after the appearance of the floral pherophylls (subtending bracts). The terminal flower is preceded by metaxyphylls and is initiated earlier than the uppermost lateral flowers of the botryoid inflorescence. Small scales (interpreted as rudimentary perianth organs) precede the stamens. These scales are more distinct in the terminal flower than in the lateral flowers. In the radially symmetrical terminal flower, small scales (or metaxyphylls) and stamens are initiated in a spiral during early development. At anthesis, stamen phyllotaxis appears irregular or approximately whorled as a result of the rapid elongation and irregular slight curvature of the stamen filaments which distorts the originally regular pattern. Finally, the numerous carpels arise simultaneously in a single whorl. It takes about 9 months for flowers to develop and the 2-year reproductive cycle of T. aralioides is typical of many trees. The floral development of T. aralioides is compared with that of other basal eudicots. The bottle-shaped, unicellular stigmatic papillae and long, decurrent stigma of basally united carpels are similar to those of the Buxales¸ suggesting a close relationship.     

Unusual alternations of floral organs in <Emphasis Type="Italic">Paeonia</Emphasis>: Structure and possible mechanism of formation

Morphological analysis of flowers was carried out in Paeonia L. cultivars. Some unusual alternations of floral organs were described: sepal-(petal-stamen) × n-carpel, where 2 ≤ n ≤ 4 (appearance of an additional zone of petal and stamen formation in the medial flower part). The identity of floral organs was not affected in the flowers with this unusual alternation. It was shown on the basis of mathematical simulation of the genes responsible for flower development that these alternations may be determined by increased pool of stem cells, which may lead to delayed termination of flower development.     

Floral development of the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>: ontogeny studies as a tool to better characterize homeotic mutations

This work provides new evidence of the complex genetic regulation necessary to accomplish flower development in legumes. Using scanning electron microscopy (SEM) analysis, we have characterized the early developmental events of the wild type Medicago truncatula flower and selected morphological characters as markers to break it down into eight different developmental stages. The order of floral organ initiation in M. truncatula and pea (Pisum sativum L.), in contrast to Arabidopsis and Antirrhinum, is unidirectional in all whorls starting from the abaxial position of the flower with a high degree of overlap. Another main difference is the existence of four common primordia from which petals and stamens differentiate. The formation of common primordia, as opposed to discrete petal and stamen primordia, has been described in many legume and non-legume plants. The main differences between pea and M. truncatula floral ontogeny are in carpel and fruit development. We also used these morphological markers as tools to characterize early alterations in the flower development of a male-sterile M. truncatula floral homeotic mutant named mtapetala. This mutant displays a phenotype resembling those of weak class B mutants with homeotic conversions of floral organ whorls 2 and 3 into sepaloid and carpelloid structures, respectively. Ontogeny studies of the mtapetala mutant flowers showed similarities with the effects of previously described loss-of-B-function mutations. Differences between ontogeny of wild type and mtapetala flowers could not be detected during the first stages (1-5) of flower development. In late stage 5, abnormal-shaped petals with acute lobes and trichomes as well as abnormal-shaped stamens were visible in whorls 2 and 3. At stage 6, the morphology of petals began to change, developing enlarged sepaloid structures bearing trichomes and first the antesepalous stamens and then the antepetalous stamens began to differentiate carpelloid anthers from filaments. Third whorl organs presented different degrees of carpelloidy. The present study should provide tools for the characterization and comparative analyses of new Medicago floral homeotic mutants and could be useful in elucidating how floral organ identity functions work in legumes.     

Evolution of flower shape in <Emphasis Type="Italic">Plantago lanceolata</Emphasis>

Plantago lanceolata produces small actinomorphic (radially symmetric), wind-pollinated flowers that have evolved from a zygomorphic, biotically pollinated ancestral state. To understand the developmental mechanisms that might underlie this change in flower shape, and associated change in pollination syndrome, we analyzed the role of CYC-like genes in P. lanceolata. Related zygomorphic species have two CYC-like genes that are expressed asymmetrically in the dorsal region of young floral meristems and in developing flowers, where they affect the rate of development of dorsal petals and stamens. Plantago has a single CYC-like gene (PlCYC) that is not expressed in early floral meristems and there is no apparent asymmetry in the pattern of PlCYC expression during later flower development. Thus, the evolution of actinomorphy in Plantago correlates with loss of dorsal-specific CYC-like gene function. PlCYC is expressed in the inflorescence stem, in pedicels, and relatively late in stamen development, suggesting a novel role for PlCYC in compacting the inflorescence and retarding stamen elongation in this wind pollinated species.     

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 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.     

Morphogenesis and Gene Mapping of <Emphasis Type="Italic">deformed interior floral organ 1</Emphasis> (<Emphasis Type="Italic">difo1</Emphasis>), a Novel Mutant Associated with Floral Organ Development in Rice

Floral organ identity and specific number directly affect anthesis habits, fertilization and grain yield. Here, we identified a deformed interior floral organ 1 (difo1) mutant from selfing progenies of indica cv. Zhonghui8015 (Zh8015) after ⁶⁰Co γ-ray treatment. Compared with the Zh8015 spikelet, the interior floral organs of the difo1 mutant present various numbers of stamens and stigmas, with no typical filament and no mature pollen grains. Most difo1 flowers exhibited an increased number of stigmas that were attached to the stamens and an intumescent ovule-like cell mass in addition to the ovary. Transverse sections of spikelets and scanning electron microscopy analysis revealed an indeterminate number of interior floral organs and abnormal early spikelet development for the difo1 mutant. Instead of the linear-shaped surface of wild-type stamens, difo1 displayed a glossy stamen surface resulting in immature stamens and complete sterility. In addition, the difo1 mutant exhibited delayed anthesis, rapid anthesis and non-extended stamens compared with wild type. Genetic analysis and gene mapping revealed that difo1 was controlled by a single recessive gene, which was fine-mapped to a 54-kb interval on the short arm of chromosome 4 between markers S22 and RM16439 harboring nine ORFs. Sequence analysis revealed that the mutant carried a single nucleotide deletion in its promoter region, which likely corresponded to the phenotype, in a C₂H₂-type zinc finger protein gene (LOC_Os04g08600). Moreover, qRT-PCR analysis showed a significantly down-regulated expression pattern for DIFO1 and many floral organ identity genes in the interior floral organs of difo1. DIFO1 is therefore an important floral organ development gene in rice, particularly with regard to interior organ meristem identity and floret primordium differentiation.     

Evolutionary correlation between floral monosymmetry and corolla pigmentation patterns in <Emphasis Type="Italic">Rhododendron</Emphasis>

Floral symmetry and pigmentation are features of flowers that are believed to be associated due to their shared influence on pollinator behaviour. However, the evolution of such associations has so far not been examined. We analysed variation in Rhododendron flowers, in a phylogenetic context, to test whether the evolution of floral symmetry types and pigment patterns are correlated. Variation in floral symmetry due to variation in corolla form, stamen flexion, stamen arrangement, pistil flexion, as well as corolla pigment patterns was documented in 98 species of Rhododendron. Phylogenetic relations among these species were estimated using maximum likelihood (ML) and Bayesian methods, building on a published molecular dataset of sequences of RNA Polymerase II subunit (RPB2-I). Evolution of the floral traits was studied using phylogenetic correlation tests and ancestral state reconstructions (maximum parsimony, MP and ML methods). Significant correlations were found between corolla pigment pattern and type of floral symmetry at the level of corolla form, stamen flexion or arrangement, and pistil flexion. As expected from their similar roles in enhancing attractability to pollinator, monosymmetric corollas and presence of pigment pattern are correlated; in addition, monosymmetry involving other whorls too shows such a relationship with pigment patterns, and with each other. Multiple evolutionary shifts were detected between monosymmetry and polysymmetry of floral traits in Rhododendron. The relationship between floral monosymmetry attributes and presence of corolla pigment patterns, and additionally, frequent evolutionary shifts in these traits suggest pollinator-mediated selective pressures in Rhododendron.     

Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism

Background  

Evolution of unisexual flowers entails one of the most extreme changes in plant development. Cultivated spinach, Spinacia oleracea L., is uniquely suited for the study of unisexual flower development as it is dioecious and it achieves unisexually by the absence of organ development, rather than by organ abortion or suppression. Male staminate flowers lack fourth whorl primordia and female pistillate flowers lack third whorl primordia. Based on theoretical considerations, early inflorescence or floral organ identity genes would likely be directly involved in sex-determination in those species in which organ initiation rather than organ maturation is regulated. In this study, we tested the hypothesis that sexual dimorphism occurs through the regulation of B class floral organ gene expression by experimentally knocking down gene expression by viral induced gene silencing.     

<Emphasis Type="Italic">APETALA2</Emphasis> like genes from <Emphasis Type="Italic">Picea abies</Emphasis> show functional similarities to their Arabidopsis homologues

In angiosperm flower development the identity of the floral organs is determined by the A, B and C factors. Here we present the characterisation of three homologues of the A class gene APETALA2 (AP2) from the conifer Picea abies (Norway spruce), Picea abies APETALA2 LIKE1 (PaAP2L1), PaAP2L2 and PaAP2L3. Similar to AP2 these genes contain sequence motifs complementary to miRNA172 that has been shown to regulate AP2 in Arabidopsis. The genes display distinct expression patterns during plant development; in the female-cone bud PaAP2L1 and PaAP2L3 are expressed in the seed-bearing ovuliferous scale in a pattern complementary to each other, and overlapping with the expression of the C class-related gene DAL2. To study the function of PaAP2L1 and PaAP2L2 the genes were expressed in Arabidopsis. The transgenic PaAP2L2 plants were stunted and flowered later than control plants. Flowers were indeterminate and produced an excess of floral organs most severely in the two inner whorls, associated with an ectopic expression of the meristem-regulating gene WUSCHEL. No homeotic changes in floral-organ identities occurred, but in the ap2-1 mutant background PaAP2L2 was able to promote petal identity, indicating that the spruce AP2 gene has the capacity to substitute for an A class gene in Arabidopsis. In spite of the long evolutionary distance between angiosperms and gymnosperms and the fact that gymnosperms lack structures homologous to sepals and petals our data supports a functional conservation of AP2 genes among the seed plants.