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.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

Molecular evolution of cycloidea-like genes in Fabaceae

The cycloidea (CYC) gene controls floral symmetry in snapdragon (Antirrhinum majus). We investigated the evolution of CYC-like genes in some species of legumes that have zygomorphic flowers. Two to four CYC-like genes were isolated from a single species. The results of NJ and ML analyses indicate that CYC-like genes in legumes group into two monophyletic clades; one group consists of eight CYC-like genes (Clade 1) and the other contains three CYC-like genes and TB1 of maize (Clade 2). These phylogenetic trees and the Shimodaira–Hasegawa test suggest that Clade 1 is a sister of the original CYC group (Clade 3). Moreover, the result of the GeneTree analysis showed that the CYC-like genes experienced repeated duplication events during the evolution of legumes. We herein speculate as to the role of CYC-like genes in legumes and discuss the evolutionary processes that these genes have undergone. Current address (Jun Yokoyama and Masayuki Maki): Division of Ecology and Evolutionary Biology, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan     

Expression pattern of CYC‐like genes relating to a dorsalized actinomorphic flower in Tengia (Gesneriaceae)

Abstract Tengia has been called a “natural peloria” in the family Gesneriaceae because it exhibits an almost perfect actinomorphic flower from whorl one to whorl three. It would be especially interesting to know whether or how CYC‐like gene activities are related to this type of perfect actinomorphic flower. To address this, we have isolated four CYC‐like TCP genes and carried out an investigation on their expression patterns in Tengia. TsCYC1C and TsCYC1D have similar expression patterns with strong signals being detected in all five petals and stamens, whereas TsCYC2A and TsCYC2B are only transiently expressed in the very early floral meristem. Our results suggest that the expansion of the expressions of TsCYC1C and TsCYC1D from the dorsal to the ventral petals is likely responsible for the evolutionary formation of the fully dorsalized actinomorphic corolla, that is, an expanded functional domain of CYC‐like gene dorsal identity in Tengia corolla. However, the expressions of TsCYC1C and TsCYC1D are not correlated with stamen abortion; therefore, TsCYC genes do not functionally repress the stamen development in Tengia flowers. This is probably due to changed cis‐activities that result in the cell cycle‐related genes uncoupling from the TsCYC regulatory pathway in Tengia.     

INFLORESCENCE AND FLORAL DEVELOPMENT IN HOUTTUYNIA CORDATA (SAURURACEAE)

The inflorescence of Houttuynia cordata produces 45–70 sessile bracteate flowers in acropetal succession. The inflorescence apical meristem has a mantle-core configuration and produces “common” or uncommitted primordia, each of which bifurcates to form a floral apex above, a bract primordium below. This pattern of organogenesis is similar to that in another saururaceous plant, Saururus cernuus. Exceptions to this unusual development, however, occur in H. cordata at the beginning of inflorescence activity when four to eight petaloid bract primordia are initiated before the initiation of floral apices in their axils. “Common” primordia also are lacking toward the cessation of inflorescence apical activity in H. cordata when primordia become bracts which may precede the initiation of an axillary floral apex. Many of these last-formed bracts are sterile. The inflorescence terminates with maturation of the meristem as an apical residuum. No terminal flowers or terminal gynoecia were found, although subterminal gynoecia or flowers in subterminal position may overtop the actual apex and obscure it. Individual flowers have a tricarpellate syncarpous gynoecium and three stamens adnate to the carpels; petals and sepals are lacking. The order of succession of organs is: two lateral stamens, median stamen, two lateral carpels, median carpel. The three carpel primordia almost immediately are elevated as part of a gynoecial ring by zonal growth of the receptacle below the attachment of the carpels. The same growth elevates the stamen bases so that they appear adnate to the carpels. The trimerous condition in Houttuynia is the result of paired or solitary initiations rather than trimerous whorls. Symmetry is bilateral and zygomorphic rather than radial. No evidence of spiral arrangement in the flower was found.     

Temporal,but not spatial,changes in expression patterns of petal identity genes are associated with loss of papillate conical cells and the shift to bird pollination in Macaronesian Lotus (Leguminosae)

• In the generally bee‐pollinated genus Lotus a group of four species have evolved bird‐pollinated flowers. The floral changes in these species include altered petal orientation, shape and texture. In Lotus these characters are associated with dorsiventral petal identity, suggesting that shifts in the expression of dorsal identity genes may be involved in the evolution of bird pollination. Of particular interest is Lotus japonicus CYCLOIDEA 2 (LjCYC2), known to determine the presence of papillate conical cells on the dorsal petal in L. japonicus. Bird‐pollinated species are unusual in not having papillate conical cells on the dorsal petal.
• Using RT‐PCR at various stages of flower development, we determined the timing of expression in all petal types for the three putative petal identity genes (CYC‐like genes) in different species with contrasting floral morphology and pollination syndromes.
• In bird‐pollinated species the dorsal identity gene, LjCYC2, is not expressed at the floral stage when papillate conical cells are normally differentiating in bee‐pollinated species. In contrast, in bee‐pollinated species, LjCYC2 is expressed during conical cell development.
• Changes in the timing of expression of the above two genes are associated with modifications in petal growth and lateralisation of the dorsal and ventral petals in the bird‐pollinated species. This study indicates that changes in the timing, rather than spatial distribution, of expression likely contribute to the modifications of petal micromorphology and petal size during the transition from bee to bird pollination in Macaronesian Lotus species.

     

Activation of the <Emphasis Type="Italic">WUS</Emphasis> gene induces ectopic initiation of floral meristems on mature stem surface in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.     

HETEROMORPHIC FLOWER DEVELOPMENT IN NEPTUNIA PUBESCENS,A MIMOSOID LEGUME

The characteristic of heteromorphic inflorescences in some mimosoid legumes such as Neptunia is a puzzling one which can be approached developmentally. Each spicate inflorescence of Neptunia pubescens includes three types of flowers: perfect in the upper half, functionally male just below the middle, and sterile or neuter at the base. Developmental studies of the inflorescence show that order of initiation of bracts on the inflorescence is acropetal, but that order of subsequent development of flowers is both acropetal and basipetal on the axis. Bract growth and initiation of the axillary floral apices at the base are inhibited or retarded, while those in the middle and upper levels continue development without interruption. The three types of floral primordia are similar during initiatory stages of organ formation and through early development. At mid-development, differences arise in floral symmetry, petal form, stamen form, and size and shape of the carpel. The functionally male flowers become strongly dorsiventral and zygomorphic while the other two morphs remain actinomorphic or nearly so. Heteromorphy arises from a combination of early suppression of organogeny plus mid-stage innovations of zygomorphy and lateral expansion of stamen primordia. These divergent developmental pathways in one inflorescence can be interpreted in part using Gould's concept of heterochrony: changes in timing of developmental events to produce different structures. Other changes in Neptunia cannot be explained by this concept, however; such changes as omission of processes (i.e., meiosis) in some organs, or addition of processes not normally present (i.e., blade formation in stamen primordia which become staminodia). It is becoming evident from work on this and other legume flowers that actual loss of organs is rare, compared to initiation followed by suppression or modification.     

Reproductive conflicts ofPalicourea padifolia (Rubiaceae) a distylous shrub of a tropical cloud forest in Mexico

We studied a population of the distylousPalicourea padifolia (Rubiaceae) in a cloud forest remnant near Xalapa City, Veracruz, México to explore possible asymmetries between floral morphs in the attractiveness to pollinators, seed dispersers, nectar robbers, floral parasites, and herbivores. We first assessed heterostyly and reciprocal herkogamy by measuring floral attributes such as corolla length (buds and open flowers), style and anther heights, stigma and stamen lengths and the distance between the anther tip to the stigma lobe. We then estimated floral and fruit attributes such as flower size, anther height, number and size of pollen grains, fruit size, seed size, nectar production, and flower and fruit standing crops to assess differences between floral morphs in attracting and effectively using mutualistic pollinators and seed dispersers. Also, floral parasitism and nectar robbing were assessed in this study as a measure of flower attractiveness to antagonists. The system seems to conform well to classical heterostyly (e.g. reciprocal stamen/style lengths, pollen and anther dimorphism, intramorph incompatibility) yet, there were several tantalizing differences observed between pin and thrum morphs. Thrum flowers have longer corollas and larger but fewer pollen grains than pin flowers. Both morphs produced the same total number of inflorescences, developed the same number of buds, and opened the same number of flowers per inflorescence during the flowering season. Nectar production and sugar concentration were similar between floral morphs but the reward was not offered symmetrically to floral visitors throughout the day. Nectar concentration was higher in pin flowers in the afternoon. The numbers of developing, fully developed, and ripe fruits were the same between floral morphs, however, fruits and seeds were larger than those of thrums. The incidence of fly larvae was higher among thrum flowers and damage by nectar robbing was the same between floral morphs. Fruit abortion patterns of flowers manually pollinated suggest intra-morph sterility (self and intramorph incompatibility). There were no differences between morphs in fruit and seed set per flower following legitimate pollination although thrums were more leaky than the pins (intramorph compatibility).     

Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development

In Arabidopsis floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT-FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT-FD and AGAMOUS-LIKE 24 (AGL24)-SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development.     

Arabidopsis inflorescence architecture requires the activities of KNOX-BELL homeodomain heterodimers

In flowering plants, post-embryonic development is mediated by the activity of shoot and root apical meristems. Shoot architecture results from activity of the shoot apical meristem (SAM), which initiates primordia, including leaves, internodes and axillary meristems, repetitively from its flanks. Axillary meristems can develop into secondary shoots or flowers. In Arabidopsis, two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), expressed in the SAM, encode DNA-binding proteins that are essential for specifying floral primordia and establishing early internode patterning events during inflorescence development. Biochemical studies show that PNY associates with the knotted1-like homeobox (KNOX) proteins, SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). PNY-BP heterodimers are essential for establishing early internode patterning events, while PNY-STM heterodimers are critical for SAM function. In this report, we examined the role of PNY, PNF and STM during development. First, we show that PNF interacts with STM and BP indicating that PNY and PNF are redundant functioning proteins. Inflorescence development, but not vegetative development, is sensitive to the dosage levels of PNY, PNF and STM. Characterization of stm-10, a weak allele in the Columbia ecotype, indicates that STM is also involved in floral specification and internode development. Our examination of the genetic requirements for PNY, PNF and STM demonstrates that these KNOX–BELL heterodimers control floral specification, internode patterning and the maintenance of boundaries between initiating floral primordia and the inflorescence meristem.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

DFL, a FLORICAULA/LEAFY homologue gene from Dendranthema lavandulifolium is expressed both in the vegetative and reproductive tissues

FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species. The inflorescence organization of chrysanthemum differs from typical dicotyledons such as Arabidopsis and Antirrhinum as clear sepals are absent, and instead, a pappus, a rudimentary sepal, is formed. To understand the mechanism of reproduction of chrysanthemum at the molecular level, DFL, a FLORICAULA/LEAFY homologous gene, was cloned from Dendranthema lavandulifolium, which is one of the original species of chrysanthemum. The DFL gene consists of a 1,236-bp open reading frame and encodes a putative protein of 412 amino acids, which is 63% identical to LFY and 70% to FLO. The expression patterns of DFL during the flower development were analyzed, and RT-PCR results showed that DFL was strongly expressed in the flower bud. In situ hybridization experiments showed that it is strongly expressed in the inflorescence bract, petal and stamen primordial tissues throughout the inflorescence development. Its expression signals were also detected in stems, leaf primordial tissues and developing inflorescence bracts.     

INITIATION AND DEVELOPMENT OF INFLORESCENCE AND FLOWER IN ANEMOPSIS CALIFORNICA (SAURURACEAE)

All flowers of Anemopsis californica, the most specialized taxon of the family Saururaceae, are initiated as individual primordia subtended by previously initiated bracts, in contrast to the common-primordium initiation of all flowers of Saururus cernuus and of most flowers of Houttuynia cordata. Floral symmetry is bilateral and zygomorphic, and the sequence of initiation among floral parts is paired or whorled. In A. californica, the six stamens arise as three common primordia, each of which later bifurcates to form a pair. The three common primordia occupy sites corresponding to the positions of the three stamens in H. cordata flowers. In Anemopsis, the filaments of each pair are connate. Each stamen pair is vascularized by a single bifurcating vascular bundle. The three carpels per flower are usually initiated simultaneously although there may be some variation. Adnation between stamens and carpels results from zonal growth. Downward extension of the locule, and proliferation and expansion of receptacular tissue and inflorescence cortical tissue around the locule below the bases of the carpels produce the inferior ovary. The inflorescence terminates its activity as a flattened apical residuum, surrounded by bracts subtending reduced flowers most of which have stamens only.     

COMPARATIVE STUDY OF THE FLORAL VASCULATURE IN SAURURACEAE

The floral vascular systems are compared among all six taxa of Saururaceae, including the two species of Gymnotheca which have not been studied previously. All are zygomorphic (dorsiventrally symmetrical), not radial as sometimes reported, in conformity with dorsiventral symmetry during organogenesis. Apocarpy in the two species of Saururus (with four carpels and six free stamens) is accompanied by a vascular system of four sympodia, each of which supplies a dorsal carpellary bundle, two ventral carpellary bundles, and one or two stamen traces. The level at which the ventral bundles diverge is the major difference in vasculature between the two species. The other four taxa are all syncarpous, and share some degree of stamen adnation and/or connation. The vascular systems also show varying degrees of fusion. The two species of Gymnotheca (with four carpels and six stamens) are very similar to each other; in both, the ventral traces of adjacent carpels fuse to form a placental bundle, which supplies the ovules and then splits into a pair of ventral strands. The flowers of Houttuynia cordata (with only three carpels and three adnate stamens) are sessile. Each flower is vascularized by three sympodia; the median adaxial sympodium is longer than the other two sympodia before it diverges to supply the adaxial organs. Three placental bundles also are formed in Houttuynia, but the three bundles differ in their origin. The median abaxial placental bundle diverges at the same level as the three sympodial bundles of the flower, while the other two lateral placental bundles diverge at a higher level from the median adaxial sympodium. Anemopsis californica, with an inferior ovary of three carpels, sunken in the inflorescence axis, and six stamens adnate to the carpels, has a vascular system very similar to that of Houttuynia cordata. The modular theory of floral evolution is criticized, on the bases of the known behavior of apical meristems and properties of vascular systems. The hypothesis is supported that saururaceous plants may represent a line of angiosperms which diverged very early.     

Stamen development in <Emphasis Type="Italic">Arabidopsis</Emphasis> is arrested by organ-specific overexpression of a cucumber ethylene synthesis gene <Emphasis Type="Italic">CsACO2</Emphasis>

Cucumber (Cucumis sativus L.) has served as a model to understand hormone regulation in unisexual flower development since the 1950s and the role of ethylene in promoting female flower development has been well documented. Recent studies cloned the F-locus in gynoecious lines as an additional copy of the ACC synthase (ACS) gene, which further confirmed the role of ethylene in the promotion of female cucumber flowers. However, no direct evidence was generated to demonstrate that increases in endogenous ethylene production could induce female flowers by arresting stamen development. To clarify the relationship between ethylene production and stamen development, we overexpressed the ethylene synthesis cucumber gene CsACO2 to generate transgenic Arabidopsis, driven by the organ-specific promoter P _AP3 . We found that organ-specific overexpression of CsACO2 significantly affected stamen but not carpel development, similar to that in the female flowers of cucumber. Our results suggested that increases in ethylene production directly disturb stamen development. Additionally, our study revealed that among all floral organs, stamens respond most sensitively to exogenous ethylene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

POLLINATION ADAPTATION IN PEDICULARIS LANCEOLATA

Cinematographic, stereophotographic, and visual observations have revealed that the zygomorphic, nectarless flower of Pedicularis lanceolata Michx. is pollinated in northeastern Ohio by pollen-foraging workers of three Bombus species. Foragers hang inverted from the asymmetrical corolla and remove pollen with their front and middle legs from anthers concealed by the galea. The stigma contacts pollen deposited especially on the median portion of the forager's first abdominal sterilite. Insect exclosures and pollinator collections demonstrated the obligate pollination dependence of the flower on Bombus workers. Of 152 corbicular pollen loads from workers foraging on P. lanceolata, Gentiana andrewsii, Prunella vulgaris and five composite species in the same habitat, 84.2% contained P. lanceolata pollen; 75.8% of the latter contained P. lanceolata pollen exclusively. Possible coadaptive relationships of Pedicularis flowers and pollinators are discussed.     

Fruit production and floral traits: correlated evolution in <Emphasis Type="Italic">Govenia</Emphasis> (Orchidaceae)

The most common estimate of reproductive success in orchids is usually fruit set. Factors such as resource limitation and certain floral traits may influence reproductive success in animal-pollinated plants. Correlated evolution of reproductive success vs. seven floral traits (inflorescence length, flower number, flower distribution along inflorescence, dorsal sepal length, lateral sepal length, flower color, and column position) was studied in eight species of Govenia. Taxa represented three lineages in the genus. Independent contrasts were calculated on a phylogeny inferred from chloroplast (trnL-F IGS) DNA sequences, and a correlation test and multiple regression were then performed. Two data sets were evaluated, one including all eight species and another excluding G. utriculata, which is autogamous. The historical analyses showed that there is a correlation between reproductive success and dorsal sepal length, column position, and flower number, these correlations suggest that changes in these floral traits usually accompany evolutionary shifts in reproductive success. Multiple regression tests suggest that changes in reproductive success can be explained by shifts in flower number, inflorescence length, column position and by dorsal sepal length. When phylogeny is taken into account, our analyses showed that evolutionary shifts in these floral traits were correlated with changes in reproductive success. Evolutionary correlation between reproductive success and floral traits might be explained by the natural selection of certain floral phenotypes by pollinators.