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.     

CYCLOIDEA 2 Clade Genes: Key Players in the Control of Floral Symmetry,Inflorescence Architecture,and Reproductive Organ Development

Undoubted lines of evidence point out that members of CYCLOIDEA (CYC) 2 clade are essential players to control flower symmetry and, amusingly, also are determinants of capitula architecture (pseudanthium). In several species, CYC-like genes influence the androecium patterning, but to date, the function of these genes in the development of gynoecium organs is less clear. In this review, we first reported details about floral symmetry and an overview of genes and molecular mechanisms regulating the development of zygomorphism in different angiosperm lineages (e.g., basal and core eudicots and monocots). Then, we paid emphasis on the role of CYC-like genes in the development of heterogamous inflorescence of sunflower as well as other Asteraceae and some species within the Dipsacaceae family. Helianthus annuus is particularly attractive because it represents a useful model to study the role of CYC-like genes on shaping floral corolla as well as the differentiation of reproductive organs in different flowers of pseudanthia. A special attention was reserved to inflorescence morphology mutants of sunflower (i.e., Chrysanthemoids2 and tubular ray flower) because they provide useful information on the role of CYC-like genes in the radiate capitulum evolution. Finally, we discuss data from literature to suggest that CYC-like genes are also co-opted to regulate stamen and carpel differentiation likely throughout their interaction with the cell cycle and flower organ identity genes. The recruitment of reproductive organs in ray flowers also supports the phylogenetic origin of a radiate inflorescence of sunflower from a discoid capitulum and suggests that in sterile zygomorphic ray flower primordia the latent identity to differentiate both microsporangium and macrosporangium was conserved.     

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.     

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.     

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.     

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.     

<Emphasis Type="Italic">DORNRÖSCHEN-LIKE</Emphasis>, an AP2 gene,is necessary for stamen emergence in Arabidopsis

To identify novel genes in petal and stamen development, a genetic screen was carried out for enhancers of the unusual B class mutant pistillata-5 (pi-5). In pi-5 flowers, second whorl organs develop as sepals rather than petals, but third whorl stamens are normal. One pi-5 enhancer, dornröschen-like-2 (drnl-2), results in third whorl positions developing as filamentous organs. In addition to enhancing the pi-5 phenotype, drnl-2 mutants also exhibit a phenotype in a wild-type PI background. Although stamen primordia are morphologically visible during early stages of flower development, they fail to enlarge in drnl-2 mutants. DRNL, which encodes a single AP2 domain protein, is expressed in a dynamic pattern in the embryo, seedling, and flower. Analysis of both the drnl-2 mutant phenotype and the DRNL expression pattern in flowers suggests that DRNL plays a critical role in stamen emergence in Arabidopsis.     

Functional analysis of <Emphasis Type="Italic">PI-</Emphasis>like gene in relation to flower development from bamboo (<Emphasis Type="Italic">Bambusa oldhamii</Emphasis>)

Bamboo flowering owns many unique characteristics and remains a mystery. To investigate the molecular mechanisms underlying flower development in bamboo, a petal-identity gene was identified as a PISTILLATA homologue named BoPI from Bambusa oldhamii (bamboo family). Expression analysis showed that BoPI was highly expressed in flower organs and gradually increased during flower development stage, suggesting that BoPI played an important role in flower development. Ectopic expression of BoPI in Arabidopsis caused conversion of sepals to petals. 35S::BoPI fully rescued the defective petal formation in the pi-1 mutant. BoPI could interact with BoAP3 protein in vitro. These results suggested that BoPI regulated flower development of bamboo in a similar way with PI. Besides flower organs, BoPI was also expressed in leaf and branch, which revealed that BoPI may involve in leaf and branch development. Similar to other MIKC-type gene, BoPI contained the C-terminal sequence but its function was controversial. Ectopic expression of the C-terminal deletion construct (BoPI- ΔC) in Arabidopsis converted sepals to petals; BoPI- ΔC interacted with BoAP3 on yeast two-hybrid assay, just like the full-length construct. The result implied that the C-terminal sequence may not be absolutely required for organ identity function in the context of BoPI.     

Effects of alien invasion by <Emphasis Type="Italic">Bombus terrestris</Emphasis> L. (Apidae) on the visitation patterns of native bumblebees in coastal plants in northern Japan

When alien pollinator species enter a native community of pollinators in which resource partitioning has been established, the pollination network between plants and pollinators may be modified through the interactions between the pollinators over the use of floral resources. We observed the floral-use patterns of native (Bombus hypocrita and B. deuteronymus) and alien (B. terrestris) bumblebee species in a coastal grassland in northern Japan. We analyzed the factors determining resource partitioning patterns. B. hypocrita tended to visit flowers with shallow or wide open corollas, such as Rosa rugosa, whereas B. deuteronymus visited flowers with complex or deeper corollas, such as Lathyrus japonicus. Given the wider floral preference of B. terrestris, floral use by the alien bumblebees consistently overlapped with that of native bumblebees. The visitation of B. terrestris to R. rugosa flowers was positively correlated with that of B. hypocrita. These bumblebee species frequently used similar floral resources, in part because of the large overlap in the seasonality of their foraging activity. The visitation frequency of B. deuteronymus to L. japonicus flowers was independent of the visitation frequency of other bumblebee species. The major visitation periods of the bumblebees to L. japonicus flowers reciprocally differed between B. deuteronymus and B. terrestris, suggesting phenological resource partitioning between these species. Our study suggests that phenological niche partitioning is more common in specialized flowers (L. japonicus) than in generalized flowers (R. rugosa).     

Intersexual mimicry and flowering phenology facilitate pollination in a dioecious habitat specialist species, <Emphasis Type="Italic">Myristica fatua</Emphasis> (Myristicaceae)

Myristica fatua is a dioecious specialist species restricted to the endangered, freshwater Myristica swamp forests in the Western Ghats, India. Earlier studies have alluded to pollination by deception in members of the Myristica genus, and thus we examined the pollination ecology comprising floral biology, flower production, flower visitors, and reproductive success in M. fatua and inferred the potential strategies that could permit such deception in this habitat specialist tree. Male flowers provide pollen rewards for an extended period of time while female flowers are rewardless and both sexes are visited by generalist insects, mainly by honeybees and stingless bees. Bee visits were significantly more frequent and longer on male than on female flowers as bees collected pollen from male flowers. We found that flower production patterns create a preponderance of males compared to females in the swamp populations. Using a model of honeybee color vision, we found the distance between the color loci of male and female flowers and based on minimum visual angle subtended by these flowers, we suggest that the two floral sexes cannot be discriminated by bees. Bees are likely deceived by the perceptual similarity of rewardless female flowers to pollen-offering male flowers and pollination is the consequence of foraging errors made by pollinators that encounter largely male–rarely female flower mosaics as they forage among clump-distributed M. fatua trees in the swamp habitat.     

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.     

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.     

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.     

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.     

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.