Floral Morphogenesis of Anemone rivularis Buch.-Ham. ex DC. var.flore-minore Maxim. （Ranunculaceae） with Special Emphasis on Androecium Developmental Sequence

The floral morphogenesis and androecium developmental sequence of Anemone rivularis Buch.-Ham. ex DC. var.flore-rninore Maxim. were observed under a scanning electron microscope (SEM) and by means of histological methods in order to expand our knowledge of the morphogenesis and development of the floral organs of the Ranunculaceae. The initiation of the floral elements is a centripetal spiral and the direction of the spiral is clockwise or anti-clockwise. However, the development of the androecium is highly unusual: in a longitudinal series of four stamens, the second stamen develops first from the inner to outer, then the third one, the fourth one and the first one in turn. The microsporogenesisand ant her maturation follows the same developmental sequence. The tepals are different from the bracts and the stamens in both shape and size in the early developmental stage, but there is no difference between the stamens and carpels in the early developmental stage. Therefore, we established a spatio-temporal process of the floral morphogenesis ofA. rivularis var.flore-rninore and offer another meaning of the floral diversity patterns attributed to the level of the genus.     

Anatomy and ontogeny of unisexual flowers in dioecious Woonyoungia septentrionalis （Dandy） Law （Magnoliaceae）

Woonyoungia septentrionalis （Dandy） Law is aceae. The floral morphology and structure of the species a dioecious species with unisexual flowers in Magnoliare conspicuously different from other species and are important to the study of floral phylogeny in this family. The floral anatomy and ontogeny were investigated to evaluate the systematic position of W. septentrionalis, using scanning electron microscopy and light microscopy. All of the floral organs are initiated acropetally and spirally. The carpels are of conduplicated type without the differentiation of stigma and style. The degenerated stamens in the female flowers have the same structures as the normal stamens at the earlier developmental stages, but they do not undergo successive development and eventually degenerate. The male floral apex was observed to have the remnants of carpels in a few investigated samples. As the bisexual flower features could be traced both in the male and female flowers in W. septentrionalis, it suggests that the flower sex in Magnoliaceae tends toward unisexual. As well as the unisexual flowers, the reduced tepals and carpels and concrescence of carpels conform to the specialized tendency in Magnoliaceae, which confirms the derived position of W. septentrionalis in this family. As the initiation pattern of floral parts of W. septentrionalis is very similar to other species in this family, it needs further investigation and especially comparison with species in Kmeria to evaluate the separation of Woonyoungia.     

Conserved Functions of Arabidopsis and Rice CC-Type Glutaredoxins in Flower Development and Pathogen Response

Glutaredoxins （GRXs） are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2-two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxyl floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ develop- ment and pathogen defence.     

ABCB1 and ABCB19 auxin transporters have synergistic effects on early and late Arabidopsis anther development

Arabidopsis abcb1 abcb19 double mutants defective in the auxin transporters ABCB1/PGP1 and ABCB19/PGP19 are altered in stamen elongation, anther dehiscence and pollen maturation. To assess the contribution of these transporters to stamen development we performed phenotypic, histological analyses, and in situ hybridizations on abcb1 and abcb19 single mutant fl owers. We found that pollen maturation and anther dehiscence are precocious in the abcb1 but not in the abcb19 mutant. Accordingly, endothecium ligni fication is altered only in abcb1 anthers. Both abcb1 and abcb1 abcb19 stamens also show altered early development, with asynchronous anther locules and a multilayer tapetum. DAPI staining showed that the timing of meiosis is asynchronous in abcb1 abcb19 anther locules, while only a small percentage of pollen grains are nonviable according to Alexander's staining. In agreement, TAM(TARDY ASYNCHRONOUS MEIOSIS), as well as BAM2(BARELY ANY MERISTEM)—involved in tapetal cell development—are overexpressed in abcb1 abcb19 young fl ower buds. Corre spondingly, ABCB1 and ABCB19 mRNA localization supports the observed phenotypes of abcb1 and abcb1 abcb19 mutant anthers. In conclusion, we provide evidence that auxin transport plays a signi ficant role both in early and late stamen development: ABCB1 plays a major role during anther development, while ABCB19 has a synergistic role.     

Floral organogenesis of Delavaya toxocarpa （Sapindaceae; Sapindales）

The floral organogenesis and development of Delavaya toxocarpa Franch. （Sapindaceae） were studied trader scanning electron microscope and light microscope to determine its systematic position within Sapindaceae. Flowers arise in terminal thyrses. The sepal primordia initiate in a spiral （2/5） sequence, which are not synchronous. The five petal primordia initiate almost synchronously and alternate with sepal primordia. Eight stamens initiate almost simultaneously and their differentiation precedes that of the petals. The last formed petal and one stamen initiate from a common primordium. Mature stamens curve inwards and cover the ovary in bud. The gynoecium begins as a hemispheric primordium on which two carpellary lobes arise simultaneously. Later in development a single gynocium is formed with two locules and two ovules per locule. Floral morphology suggests a closer affinity with Sapindaceae, although certain features of floral ontogenesis are similar to those observed in certain members of the former Hippocastanaceae, such as Handeliodendron.     

Gibberellin and Jasmonate Crosstalk during Stamen Development

Gibberellin （GA） and jasmonate （JA） are two types of phytohormones that play important roles during stamen development. For example, Arabidopsis plants deficient in either of GA or JA develop short stamens. An apparent question to ask is whether GA action and JA action during stamen filament development are independent of each other or are in a hierarchy. Recent studies showed that GA modulates the expression of genes essential for JA biosynthesis to promote JA production and high levels of JA will induce the expression of three MYB genes MYB21, MYB24 and MYB57. These three MYB genes are crucial factors for the normal development of stamen filament in Arabidopsis.     

Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya

The homologous genes FLORICAULA （FLO） in Antirrhinum and LEAFY （LFY） in Arabidopsis are known to regulate the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFY homologs of the two woody tree species： California sycamore （Platanus racemosa） and black cottonwood （Populus trichocarpa）. Despite the high sequence similarity within two conserved regions, the N-terminal proline-rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional LFY ortholog NEEDLY of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there is only one copy of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low level in leaf primordia, but it is expressed at a high level in the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types - male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and has limited effect on sex differentiation in papaya.     

Isolation and Characterization of a Putative Class E Gene from Taihangia rupestris

Studies In model plants showed that SEPALLATA （SEP） genes are required for the Identification of floral organs and the determination of floral meristems In Arabidopsis. In this paper a SEP homolog, TrSEP3, was Isolated from a China-specific species, Taihangla rupestrisi Yü et LI. Phylogenetlc analysis showed that the gene belongs to the SEP3-clade of SEP （previous AGL2） subfamily. In situ hybridization was used to reveal the potential functional specification, and the results showed that TrSEP3 expression was first observed in floral meristems and then confined to the floral primordla of the three inner whorls. In the matured flower, TrSEP3 was strongly expressed In the tips of pistils and weak In stamens and petals. The evolution force analysis shows that TrSEP3 might undergo a relaxed negative selection. These results suggested that TrSEP3 may not only function In determining the identity of floral merlstems and the primordia of three inner whorls, but also function In matured reproductive organs.     

Characterization of the Rice Floral Organ Number Mutant fon3

A spontaneous rice mutant named floral organ number 3 (fon3) had major mutations in floral organ numbers. Genetic analysis indicated that fort3 acted as a single recessive gene. Microscopic observation showed that the number of floral organs infon3 increased centripetally. For example, the number of pistils was the more frequently increased than organs in the outer whorls. Homeotic conversion of lodicules and glumes into palea/lemma-like organs was observed in some flowers. Scanning electron microscopy observation showed that the size of flower meristems was maintained the same or similar until the lemma primordium started to differentiate, at which time the floral meristem became enlarged, suggesting abnormal development of the inner whorls of rice florets. The relationship of fort3 with other similar rice mutants is discussed.     

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.     

Gene trap lines define domains of gene regulation in Arabidopsis petals and stamens

To identify genes involved in Arabidopsis thaliana petal and stamen organogenesis, we used a gene trap approach to examine the patterns of reporter expression at each stage of flower development of 1765 gene trap lines. In 80 lines, the reporter gene showed petal- and/or stamen-specific expression or lack of expression, or expression in distinct patterns within the petals and/or the stamens, including distinct suborgan domains of expression, such as tissue-specific lines marking epidermis and vasculature, as well as lines demarcating the proximodistal or abaxial/adaxial axes of the organs. Interestingly, reporter gene expression was typically restricted along the proximodistal axis of petals and stamens, indicating the importance of this developmental axis in patterning of gene expression domains in these organs. We identified novel domains of gene expression along the axis marking the midregion of the petals and apical and basal parts of the anthers. Most of the genes tagged in these 80 lines were identified, and their possible functions in petal and/or stamen differentiation are discussed. We also scored the floral phenotypes of the 1765 gene trap lines and recovered two mutants affecting previously uncharacterized genes. In addition to revealing common domains of gene expression, the gene trap lines reported here provide both useful markers and valuable starting points for reverse genetic analyses of the differentiation pathways in petal and stamen development.     

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 ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo.     

Stamenless, a tomato mutant with homeotic conversions in petals and stamens

A tomato (Lycopersicon esculentum Mill.) monogenic semidominant mutation, stamenless (sl), which results in homeotic conversions in two adjacent floral whorls, was studied. When grown at standard temperature, flowers of sl/sl plants showed sepaloid petals in the second whorl and strong transformation of stamens to carpels in whorl three. These transformed carpels were fused with each other and with the genuine carpels in the fourth whorl to form a unique gynoecium. The mutation is semidominant since heterozygous plants showed a phenotype intermediate between that of the wild type (WT) and that of homozygous mutant plants, with nearly WT petals but with feminized stamens bearing naked ovules on the base of their adaxial face. The initiation and position of organ primordia in sl/sl flowers were not altered when compared with WT primordia although development of organ primordia in the second and third whorls deviated from WT at an early stage as observed by scanning electron microscopy. The mutant phenotype is temperature sensitive and when sl/sl plants were cultured at low temperature, the morphology of some flowers resembled that of the WT. This reversion of the mutant phenotype is also induced by treatment of young sl/sl plants with gibberellic acid, providing evidence that gibberellin synthesis or sensitivity could mediate the effect of low temperature on the mutant phenotype. Southern blot analyses using a Deficiens-homologous gene from Solanum tuberosum as a probe showed a restriction-fragment-length polymorphism (RFLP) linked to the sl mutation. This result indicates that the mutation affects a Deficiens-like gene that controls the identity of petals and stamens. Received: 10 December 1998 / Accepted: 29 March 1999     

Ectopic expression of SUPERMAN suppresses development of petals and stamens

The floral regulatory gene SUPERMAN (SUP) encodes a C2H2 type zinc finger protein that is required for maintaining boundaries between floral organs in Arabidopsis. It has been proposed that the main function of SUP is to balance cell proliferation in the third and fourth whorl of developing flowers, thereby maintaining the boundaries between the two whorls. To gain further insight into the function of SUP, we have ectopically expressed SUP using the promoter of APETALA1 (AP1), a gene that is initially expressed throughout floral meristems and later becomes restricted to the first and second whorls. Flowers of AP1::SUP plants have fewer floral organs, consistent with an effect of SUP on cell proliferation. In addition, the AP1::SUP transgene caused the conversion of petals to sepals and suppressed the development of stamens. The expression of the B function homeotic gene APETALA3 (AP3) and its regulator UNUSUAL FLORAL ORGANS (UFO) were delayed and reduced in AP1::SUP flowers. However, SUP does not act merely through UFO, as constitutive expression of UFO did not rescue the defects in petal and stamen development in AP1::SUP flowers. Together, these results suggest that SUP has both indirect and direct effects on the expression of B function homeotic genes.     

THE DEVELOPMENTAL BASIS OF TRISTYLY IN EICHHORNIA PANICULATA (PONTEDERIACEAE)

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

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

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

Flower development in the organ number mutant clavata1-1 of Arabidopsis thaliana (Brassicaceae)

Flowers of the organ number (meristic) mutant clavata1-1 of Arabidopsis thaliana (Brassicaceae) were studied to examine timing and patterns of floral organogenesis as compared to the wild type. All clavata1-1 flowers examined had four- instead of two-loculed gynoecia; half showed increased numbers of stamens; and 10% formed increased numbers of sepals. An inflorescence plastochron index was used to establish the timing of developmental events during flower organogenesis. clavata1-1 flowers initiate faster but grow more slowly than in the wild type. The stages of sepal and stamen initiation were prolonged compared to those of the wild type. Although gynoecial initiation was not prolonged, the preceding stage was and it was characterized by a proliferation of meristematic cells above the initiating stamens. The clavata1-1 flower apex did not become wider than that of the wild type until after the establishment of the gynoecium. We propose that clavata1-1 is a heterochronic mutant, where flower organ number increases are due partly to prolongation of organ initiation stages.