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.     

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.     

The role of floral structure and biotic factors in determining the occurrence of florivorous thrips in a dystilous shrub

Elucidating the factors determining the occurrence of florivorous organisms is an essential step for comprehending arthropod–plant interactions, especially when considering florivores that use flowers/inflorescences as microhabitats. In this study, we characterize the interaction between florivorous thrips (Thysanoptera) and Palicourea rigida (Rubiaceae), a distylous hummingbird-pollinated shrub. We investigated the relative role of different factors in determining thrips occurrence in the flower and inflorescence microhabitats. Furthermore, we experimentally examined the protective role of corolla influencing thrips exploration of floral buds. Frankliniella musaeperda (Thripidae) was the only species recorded on P. rigida, feeding on floral tissue, pollen and nectar. Thrips occurrence was not related to distyly, but rather to floral stage. Open flowers presented the highest abundance of thrips, followed by senescent flowers and then buds. The experimental opening of buds translated in increased thrips occurrence, indicating that F. musaeperda manage to explore the microhabitat offered by the floral chamber, as long as there is an opening in the corolla. In inflorescences, thrips abundance was negatively related to the number of ants visiting extrafloral nectaries. We found that the marked difference between floral morphs of distylous plants is not necessarily reflected in the abundance of florivores. Thrips seek for floral cavities, preferentially those with fresh tissue, which may confer nutrient-rich food and protection. Buds also provide this; however, the enclosed petals are an effective barrier against F. musaeperda entrance. At inflorescence scale, presence of mutualistic ants in high numbers can drive away these flower-feeding insects. Despite the abundance of thrips in the flowers, there was no evidence of any functional relationship, either of pollination for flowers or of breeding for insects. We demonstrate here that in the flower/inflorescence microhabitat, structural and biotic factors play a key role in the exploitation and occupation by insect florivores.     

Functional analysis of the promoters of B-class MADS-box genes in London plane tree and their application in genetic engineering of sterility

Breeding flowerless and/or fruitless varieties are highly desirable for London plane tree because it can prevent pollen- and fruit-mediated environmental contamination. Floral tissue-specific cell ablation is an efficient method to create such sterile plants. Here we isolated and characterized APETALA3 (AP3)-like and PISTILLATA (PI)-like genes and the promoters of PaAP3 and PaPI, in London plane tree respectively. The promoter fragments were fused to GUS (β-glucuronidase) and BARNASE gene, respectively, and transformed into tobacco plants. In pPaAP3::GUS transgenic plants, the GUS activity could be detected in various organs, including leaves, stems and all floral organs. Furthermore, most tobacco plants transformed with pPaAP3::BARNASE were dead and the survivals showed abortion of inflorescence. In contrast, heterologous expression of pPaPI::GUS in tobacco plants led to specific GUS activity in the inner three whorls of flowers. Accordingly, tobacco plants transformed with pPaPI::BARNASE lack petal, stamen and pistil, with only sepal left. The results suggest that sterile lines of P. acerifolia may be obtained by genetic engineering with pPaPI::BARNASE construct, which might solve the problems of shedding fruit hairs and disseminative pollens, reducing air pollution and reducing the allergens that harmful to human health.     

Bee pollination and evidence of substitutive nectary in <Emphasis Type="Italic">Anadenanthera colubrina</Emphasis> (Leguminosae-Mimosoideae)

Anadenanthera colubrina (Vell.) Brenan (Leguminosae-Mimosoideae) is a widely-distributed tree in seasonally dry tropical forests of South America that was classified previously as lacking nectaries. However, some studies have stated that its flowers produce nectar, while others analyzed the composition of unifloral honey produced from A. colubrina flowers, raising the question about nectar production in the species. We studied the pollination and reproductive biology of A. colubrina var. cebil (Griseb.) Altschul in a natural population in the Caatinga, northeastern Brazil. Reproductive phenology, sexual system, floral biology, resource, and pollinators were investigated. We analyzed the breeding system through controlled pollinations for addressing its dependence on pollen vectors for reproduction. Anadenanthera colubrina flowered in the dry season, flower heads are heteromorphic, with staminate flowers at the base and perfect flowers at the apex of the inflorescence, characterizing andromonoecy. Anthesis is diurnal. We observed small drops of nectar at the apex of the petals of some flowers per inflorescence. Together with observations on flower visitor behavior and histochemical tests, we propose that A. colubrina produces floral nectar at the apex of the corolla, characterizing a substitutive nectary (sensu Vogel). This is the first record of substitutive nectary in the Mimosoideae and the first record of andromonoecy in the genus. Bees were the main pollinators (higher frequency), although other insects such as wasps, butterflies, and small beetles were also observed collecting nectar and/or pollen. The species is self-incompatible, thus depending on insect pollen vectors, mainly bees, for reproduction.     

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.     

Diversity and evolution of pollinator rewards and protection by <Emphasis Type="Italic">Macaranga</Emphasis> (Euphorbiaceae) bracteoles

Flowering plants have modified their floral organs in remarkably diverse ways to optimize their interaction with pollinators. Although floral organs represent a major source of floral diversity, many plants also use extrafloral organs, such as bracts and bracteoles, in interacting with pollinators; however, the evolutionary dynamics of non-floral organs involved in pollination are poorly studied. The genus Macaranga is characterized by protective mutualisms with ants that potentially interfere with pollinators on flowers. Macaranga flowers lack perianths and, notably, bracteoles serve the dual function of rewarding pollinators and protecting them from guarding ants; in one group of species, bracteoles provide a nectar reward to generalist pollinators, while in another group, bracteole “chambers” protect thrips or hemipteran pollinators that use these structures as feeding and breeding sites. We examined the diversity and evolutionary dynamics of inflorescence morphology in Macaranga, focusing on bracteoles. We recognized three inflorescence types based on examination of herbarium materials: Discoid-gland, which possess disc-shaped glands on the bracteole surfaces (including all the generalist-pollinated species); Enclosing, in which bracteoles cover flowers (including all the thrips- and hemipteran-pollinated species); and Inconspicuous, in which bracteoles are small, narrow or absent. Ancestral state reconstruction indicated that inflorescence morphologies have changed multiple times in the genus. These findings suggest that morphological changes in non-floral characters (bracteoles) of Macaranga species have occurred as frequently as in the floral structures of many flowering plants. The multiple evolutions of the Enclosing bracteoles, which protect pollinators, might have been facilitated by pollination interference from mutualistic ants.     

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.     

Relationship between seasonal progression of floral meristem development and <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> expression in the deciduous tree <Emphasis Type="Italic">Fagus crenata</Emphasis>

Estimating the timing of flower bud formation in plants is essential to identify environmental factors that regulate floral transition. The presence of winter dormancy between the initiation of flowers and anthesis, characteristic of most trees in the temperate forests, hampers accurate estimation of the timing of floral transition. To overcome this difficulty, expression levels of flowering-time genes could be used as indicators of the timing of floral transition. Here, we evaluated the usefulness of molecular markers in estimating the timing of floral transition in Fagus crenata, a deciduous tree that shows intermittent and synchronized flowering at the population level. We selected FLOWERING LOCUS T (FT) as a candidate molecular marker and quantified the expression levels of its ortholog in F. crenata (FcFT). Subsequently, we analyzed the relationship between morphogenetic changes that occur between the vegetative state of the buds and the initiation of floral organs, and compared the FcFT expression levels in reproductive and vegetative buds, collected from spring to autumn. FcFT expression in leaves peaked at least two weeks before the morphological changes associated with flowering were visible in the buds in late July. FcFT expression levels were significantly higher in the reproductive buds than in the vegetative buds in July. These results suggest that the FcFT expression in July is a reliable indicator of the timing and occurrence of floral transition. This study highlights the utility of molecular tools in unraveling reproductive dynamics in plants, in combination with ecological and physiological approaches.     

Molecular cloning and spatiotemporal expression of <Emphasis Type="BoldItalic">APETALA1</Emphasis>-like gene in <Emphasis Type="BoldItalic">Lonicera macranthoides</Emphasis>

APETALA1 (AP1), a floral meristem identity gene controls the flowering time and floral transition, and plays an important role in inflorescence and floral organ development. The full-length cDNA for AP1 was obtained by rapid amplification of the cDNA ends (RACE) so that the roles of AP1 in Lonicera macranthoides (Lm-AP1) could be better understood. AP1 (accession number in GenBank: MF418642) consisted of a 729-bp open reading frame encoding a protein that contained 242 amino acids, had a deduced molecular mass of 27.9919 kDa and a theoretical isoelectric point of 8.75. No signal peptide or transmembrane domains were detected in the sequences located in the nucleus, but it contained conserved sequences for MADS and the K-box. In the secondary structure, the \(\alpha \)-helix accounts for 60.74%, the \(\beta \)-turn 3.72%. The real-time polymerase chain reaction revealed that AP1 was more highly expressed in flowers, especially at the fourth flowering stage, which implied that it may play a role in flower development. Other L. macranthoides organs, such as stems and leaves, also expressed AP1. This research provided the basis for further analysis of the AP1 functional mechanism during L. macranthoides development.     

Why are orchid flowers so diverse? Reduction of evolutionary constraints by paralogues of class B floral homeotic genes

Background

The nearly 30 000 species of orchids produce flowers of unprecedented diversity. However, whether specific genetic mechanisms contributed to this diversity is a neglected topic and remains speculative. We recently published a theory, the ‘orchid code’, maintaining that the identity of the different perianth organs is specified by the combinatorial interaction of four DEF-like MADS-box genes with other floral homeotic genes.

Scope

Here the developmental and evolutionary implications of our theory are explored. Specifically, it is shown that all frequent floral terata, including all peloric types, can be explained by monogenic gain- or-loss-of-function mutants, changing either expression of a DEF-like or CYC-like gene. Supposed dominance or recessiveness of mutant alleles is correlated with the frequency of terata in both cultivation and nature. Our findings suggest that changes in DEF- and CYC-like genes not only underlie terata but also the natural diversity of orchid species. We argue, however, that true changes in organ identity are rare events in the evolution of orchid flowers, even though we review some likely cases.

Conclusions

The four DEF paralogues shaped floral diversity in orchids in a dramatic way by modularizing the floral perianth based on a complex series of sub- and neo-functionalization events. These genes may have eliminated constraints, so that different kinds of perianth organs could then evolve individually and thus often in dramatically different ways in response to selection by pollinators or by genetic drift. We therefore argue that floral diversity in orchids may be the result of an unprecedented developmental genetic predisposition that originated early in orchid evolution.Key words: Orchidaceae, orchid evolution, evo-devo; perianth, class B genes, DEFICIENS, subfunctionalization, neofunctionalization, gene duplication, peloria, modularization     

Floral meristem size and organ number correlation in <Emphasis Type="Italic">Eucryphia</Emphasis> (Cunoniaceae)

We present a comparative flower ontogenetic study in five species of the genus Eucryphia with the aim of testing whether differences in the organ number observed can be explained by changes in the meristematic size of floral meristem and floral organs. Species native to Oceania, viz. E. milliganii, E. lucida and E. moorei, have the smallest gynoecia with ca. 6 carpels, while the Chilean E. glutinosa and E. cordifolia present more than ten carpels. E. milliganii has the smallest flower with the lowest stamen number (ca. 50), while the other species produce around 200 stamens and more. Standardized measurements of meristematic sectors were taken in 49 developing flowers that were classified into three well-defined ontogenetic stages. Sizes of meristems varied significantly among species within each developmental stage as revealed by ANOVA analyses. Significant regressions between organ number and corresponding meristem size were consistent with the premise that a larger meristem size prior to organ initiation could be determining for a higher organ number. Flower organogenesis in Eucryphia also involves relevant meristem expansion while the organs are initiated, which results in a particular androecium patterning with a chaotic stamen arrangement. Meristem expansion also appears to be slower but more extensive in species with larger initial meristematic size, suggesting that flower phenotype can be determined in ontogeny by this heterochronic interplay of space and time.