ORGANOGENESIS IN FLOWERS OF THE HOMEOTIC GREEN PISTILLATE MUTANT OF TOMATO (LYCOPERSICON ESCULENTUM)

The flowers of a previously undescribed recessive mutant of tomato, green pistillate, show strong and consistent homeotic transformation of petals to sepals in whorl two and of stamens to carpels in whorl three. The phenotype at early and later stages is compared with wild type by scanning electron microscopy. Wild type and mutant show no difference in the pattern or timing of third whorl organ initiation, as shown by allometric analysis of scanning electron micrographs of early stages. This confirms that the mechanisms governing organ identity are distinct from those governing the positions and numbers of organs initiated; the former can be altered without changes in the latter. Mutant and wild type organs are compared by allometric analysis of dimensions of flowers dissected throughout development. The sepaloid petals (whorl 2) and the carpelloid stamens (whorl 3) in the mutant elongate at relative rates normal for the wild type organ of the whorls they occupy. This suggests that some aspects of organ growth, such as elongation rate, may also be independent of mechanisms governing organ identity.     

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.     

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus

Although much progress has been made in understanding how floral organ identity is determined during the floral development, less is known about how floral organ is elaborated in the late floral developmental stages. Here we describe a novel floral mutant, wrinkled petals and stamens1 （wps1）, which shows defects in the development of petals and stamens. Genetic analysis indicates that wpsl mutant is corresponding to a single recessive locus at the long arm of chromosome 3. The early development of floral organs in wpsl mutant is similar to that in wild type, and the malfunction of the mutant commences in late developmental stages, displaying a defect on the appearance of petals and stamens. In the mature flower, petals and stamen filaments in the mutant are wrinkled or folded, and the cellular morphology under L1 layer of petals and stamen filaments is abnormal. It is found that the expression patterns of floral organ identity genes are not affected in wpsl mutants compared with that of wild type, consistent with the unaltered development of all floral organs. Furthermore, the identities of epidermal cells in different type of petals are maintained. The histological analysis shows that in wpsl flowers all petals are irregularly folded, and there are knotted structures in the petals, while the shape and arrangement of inner cells are malformed and unorganized. Based on these results, we propose that Wpsl acts downstream to the class B floral organ identity genes, and functions to modulate the cellular differentiation during the late flower developmental stages.     

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.     

DIFFERENTIAL SENSITIVITY OF “DOUBLE” AND “SINGLE” FLOWERS OF NIGELLA DAMASCENA (RANUNCULACEAE) TO EMASCULATION AND TO GA3

In Nigella damascena “double” flower is inherited as a single-gene recessive to “single” flower. Early emasculation of “double” flowers greatly inhibits gynoecium development and, to a lesser degree, development of sepals and bracts. No comparable inhibition of gynoecia was induced in “single” flowers though some sepal and bract inhibition was detected. A differential nutritional requirement for organ initiation on cultured flower apices was also detected. Apices of both varieties initiated stamens and carpels if kinetin was added to the basal medium. However, in the absence of kinetin, GA₃ was required for organ initiation in “doubles” but completely inhibited stamen initiation in “singles.” Both these differential effects are thought to reflect rather different patterns of gibberellin metabolism in the two genetic strains.     

Role of phytochrome B in organ formation processes in Cucumis sativus L.

The role of phytochrome B in the organogenesis process in the apical and axillary shoot meristems during early ontogenesis stages in cucumber Cucumis sativus L. at photoperiods (day/night) 10/14, 16/8 h, and continuous light in comparison with wild type plants and phytochrome B-deficient mutant (lh-mutant) was investigated. In mutant phytochrome B, deficiency caused inhibition of initiation of leaves both in the main shoot and lateral shoots and increased the number of flower buds (IV stage of organogenesis). With continuous light, the number of lateral shoots and flowers during stage IV of organogenesis in wild-type plants increased twofold in comparison with the mutant. Short-term temperature drops did not induce floral ontogenesis in mutants but increased the number of off-shoots in both experimental variants during a long photoperiod and continuous lighting. We propose that phytochrome B, by increasing the compactness of chromatin, may facilitate coordination of ontogenesis processes with changing environmental conditions.     

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG'mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.     

Influence of the ABRUPTUS/PINOID gene on the expression of homeotic mutations apetala 3-1 and pistillata-1 in Arabidopsis Thaliana (L.) Heynh.

Results of the flower structure analysis of single mutants abr, ap3-1, and pi-1 and double mutants abr ap3-1 and abr pi-1 of the Arabidopsis thaliana are presented. An increase in the expressivity of the ap3-1 and pi-1 mutations against a background of the abr mutation which break auxin transport was detected. Unlike flowers of parental mutant plants which had stamens in our experiment, stamens were absent in basal flowers of double mutants. It can be assumed that anomalies in auxin distributions in cells can break the program of the development of stamens which is triggered by the ap3-1 and pi-1 alleles remaining the residual function during the plant growing at 21–24°C.     

Development of male and female flower in Asparagus officinalis. Search for point of transition from hermaphroditic to unisexual developmental pathway

Asparagus officinalis is a dioecious plant. The flowers start to develop as hermaphrodites and later become unisexual. In female flowers the stamens degenerate, while in male flowers the ovary stops growing without degenerating. We have examined young asparagus flowers using SEM and optical microscopy in order to determine the exact moment of transition from hermaphroditic to unisexual development. We defined 13 stages of development, starting from flower primordia up to completely mature flowers and labelled them with numbers from -6 to 7. The first five stages are fully hermaphroditic: a difference between sexes becomes visible at stage — 1 when the style begins to develop in female flowers. Degeneration of stamens in female flowers starts somewhat later. At the stage of transition, some differences between sexes also appear in the bidimensional polypeptide pattern of flowers. RNase activity shows a distinct peak at this stage (in female flowers only), probably related to stamen degeneration.     

Mutations associated with floral organ number in rice

How floral organ number is specified is an interesting subject and has been intensively studied in Arabidopsis thaliana. In rice (Oryza sativa L.), mutations associated with floral organ number have been identified. In three mutants of rice, floral organ number 1 (fon1) and the two alleles, floral organ number 2-1 (fon2-1) and floral organ number 2-2 (fon2-2), the floral organs were increased in number centripetally. Lodicules, homologous to petals, were rarely affected, and stamens were frequently increased from six to seven or eight. Of all the floral organs the number of pistils was the most frequently increased. Among the mutants, fon1 showed a different spectrum of organ number from fon2 -1 and fon2 -2. Lodicules were the most frequently affected in fon1, but pistils of more than half of fon1 flowers were unaffected; in contrast, the pistils of most flowers were increased in fon2 -1 and fon2-2. Homeotic conversion of organ identity was also detected at a low frequency in ectopically formed lodicules and stamens. Lodicules and stamens were partially converted into anthers and stigmas, respectively. Concomitant with the increased number of floral organs, each mutant had an enlarged apical meristem. Although meristem size was comparable among the three mutants and wild type in the early phase of flower development, a significant difference became apparent after the lemma primordium had differentiated. In these mutants, the size of the shoot apical meristem in the embryo and in the vegetative phase was not affected, and no phenotypic abnormalities were detected. These results do not coincide with those for Arabidopsis in which clavatal affects the sizes of both shoot and floral meristems, leading to abnormal phyllotaxis, inflorescence fasciation and increased floral organs. Accordingly, it is considered that FON1 and FON2 function exclusively in the regulation of the floral meristem, not of the vegetative meristem.Abbreviation DIC differential interference contrast This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.     

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     

Comparative floral development in male and female plants of Palmer amaranth (Amaranthus palmeri)

Premise

Characterizing the developmental processes in the transition from hermaphroditism to unisexuality is crucial for understanding floral evolution. Amaranthus palmeri, one of the most devastating weeds in the United States, is an emerging model system for studying a dioecious breeding system and understanding the biological traits of this invasive weed. The objectives of this study were to characterize phases of flower development in A. palmeri and compare organogenesis of flower development in female and male plants.

Methods

Flower buds from male and female plants were dissected for light microscopy. Segments of male and female inflorescences at different stages of development were cut longitudinally and visualized using scanning electron microscopy.

Results

Pistillate flowers have two to three styles, one ovary with one ovule, and five obtuse tepals. Staminate flowers have five stamens with five acute tepals. Floral development was classified into 10 stages. The distinction between the two flower types became apparent at stage four by the formation of stamen primordia in staminate flowers, which developed female and male reproductive organs initially, as contrasted to pistillate flowers, which produced carpel primordia only. In staminate flowers, the putative carpel primordia changed little in size and remained undeveloped.

Conclusions

Timing of inappropriate organ termination varies across the two sexes in A. palmeri. Our study suggests that the evolution of A. palmeri from a cosexual ancestral state to complete dioecy is still in progress since males exhibited transient hermaphroditism and females produced strictly pistillate flowers.     

HELICAL FLORAL ORGANOGENESIS IN GLEDITSIA,A PRIMITIVE CAESALPINIOID LEGUME

In order to determine the extent of floral ontogenetic differences among species of a genus, six species of Gleditsia were studied. Gledilsia is one of only two leguminous genera known in which there is completely helical succession of floral organs. Floral ontogeny was compared in three species (Gleditsia amorphoides, G. aquatica, and G. triacanthos), and late stages in six species (including the first three plus G. caspica, G. delavayi, and G. japonica). Other unusual primitive developmental features include the unequal-sized flower primordia which produce flowers of variable merosity. Order of floral development is also loosely controlled, so that flowers of different growth stages are intermixed in the inflorescence. Variable features include the occurrence of floral bracts, merosity of flowers, number of organs, and position of the first organ (sepal) initiated. The inflorescence type, while usually a raceme, often has lateral branches near the base, or fascicles of flowers at some points. A terminal flower often is present, although not in all species. Sex of flowers and inflorescences also varies, although floral initiation tends to include both stamens and carpel primordia. Suppression of one or the other may occur at different stages of development. Carpel orientation also varies; the cleft may be tilted or inverted occasionally. It is proposed that absence of subtending floral bracts influences development so as to favor radial symmetry and establishment of other “chaotic” characters seen in Gledilsia flowers.     

Analyses of the floral organ morphogenesis and the differentially expressed genes of an apetalous flower mutant in <Emphasis Type="Italic">Brassica napus</Emphasis>

The floral organ morphogenesis of the apetalous flower mutant Apet33-10 in Brassica napus was investigated and the result showed that all the floral organ morphogenesis was normal except that petal primordium was not observed during flower development. Eighteen genes were found to be down regulated in early floral buds (less than 200 μm in length) of Apet33-10 at the stage of floral organ initiation by means of suppressive subtraction hybridization (SSH) and RT-PCR. These genes were involved in petal identity, calcium iron signal transduction, mRNA processing, protein synthesis and degradation, construction of cytoskeleton, hydrogen transportation, nucleic acid binding, alkaloid biosynthesis and unknown function. Three overall coding region cDNAs of APETALA3 (AP3) gene, BnAP3-2, BnAP3-3 and BnAP3-4 were obtained by RT-PCR, respectively. Real-time quantitative PCR analysis showed that the expression ratio among BnAP3-2, BnAP3-3 and BnAP3-4 was 3.67:3.68:1 in early floral buds of wild type Pet33-10. The expression level of BnAP3-2, BnAP3-3 and BnAP3-4 in early floral buds of Apet33-10 was down-regulated to 36.6, 28.3 and 66.8% with the comparison of that of wild type, respectively, and the overall expression level of AP3 genes in apetalous mutant amounted to 45.0% of that in wild type. The difference in the expression level of each AP3 gene in stamen between apetalous and wild type lines was not significant. It is suggested that lower abundant expression of AP3 genes during the early flower development might be enough for stamen primordium initiation, but not enough for petal primordium initiation in the apetalous line Apet33-10. Y.T. Zhou and H.Y. Wang are committed as the first author.     

Ethylene perception is involved in female cucumber flower development

It is well established that ethylene promotes female flower development in cucumber. However, little is known about how the gaseous hormone selectively affects female flowers, and what mechanism it uses. Previously, we found organ‐specific DNA damage in the primordial anther of female cucumber flowers. This finding led to a hypothesis that ethylene might promote female flower development via the organ‐specific induction of DNA damage in primordial anthers. In this study, we tested this hypothesis first by demonstrating ethylene induction of DNA damage via the ethylene signaling pathway using cucumber protoplasts. Then, using representative component genes of the ethylene signaling pathway as probes, we found that one of the ethylene receptors, CsETR1, was temporally and spatially downregulated in the stamens of stage‐6 female cucumber flowers, especially along with the increase of the nodes. Furthermore, by constructing transgenic Arabidopsis plants with organ‐specific expression of antisense CsETR1 under the control of an AP3 promoter to downregulate ETR1 expression in the stamens, we generated Arabidopsis ‘female flowers’, in which the abnormal stamens mimic those of female cucumber flowers. Our data suggest that ethylene perception is involved in the arrest of stamen development in female cucumber flowers through the induction of DNA damage. This opens up a novel perspective and approach to solve the half‐century‐long puzzle of how gaseous ethylene selectively promotes female flowers in the monoecious cucumber plant.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

Pistillate and staminate flower development in dioecious Pistacia vera (Anacardiaceae)

The development of staminate and pistillate flowers in the dioecious tree species Pistacia vera L. (Anacardiaceae) was studied by scanning electron microscopy with the objective of determining organogenetic patterns and phenology of floral differentiation. Flower primordia are initiated similarly in trees of both sexes. Stamen and carpel primordia are initiated in both male and female flowers, and the phenology of organ initiation is essentially identical for flowers of both sexes. Vestigial stamen primordia arise at the flanks of pistillate flower apices at the same time functional stamens are initiated in the staminate flowers. Similarly, a vestigial carpel is initiated in staminate flowers at the same time the primary, functional carpel is initiated in pistillate flower primordia. Differences between the two sexes become apparent early in development as, in both cases, development of organs of the opposite sex becomes arrested at the primordial stage. Male flowers produce between four and six mature functional stamens and female flowers produce a gynoecium with one functional and two sterile carpels.     

濒危植物庙台槭生殖生物学研究1.花序和花的形态及发育

庙台械的花序为有限花序,由一顶花和6—9枝侧花枝组成,属圆锥状聚伞花序。一个花序共有14—29朵花,包括两性花、雄花和无性花三类花。根据花在花序上着生的位置,可分为三级。7月初,花序原基形成,在花序轴伸长的同时,侧面形成侧花枝轴原基。花序的顶花最早进行个体发育,随后是侧花枝顶花;侧花枝上同一级花的发育顺序则是从花序的下面向上进行。花器官发生时,花萼原基最先形成,然后是花瓣、雄蕊、心皮和胚珠。     

The mutants compacta ?hnlich, Nitida and Grandiflora define developmental compartments and a compensation mechanism in floral development in Antirrhinum majus

In order to improve our understanding of floral size control we characterised three mutants of Antirrhinum majus with different macroscopic floral phenotypes. The recessive mutant compacta ?hnlich has smaller flowers affected mainly in petal lobe expansion, the dominant mutant Grandiflora has overall larger organs, whilst the semidominant mutation Nitida exhibits smaller flowers in a dose-dependent manner. We developed a cell map in order to establish the cellular phenotypes of the mutants. Changes in organ size were both organ- and region-specific. Nitida and compacta ?hnlich affected cell expansion in proximal and distal petal regions, respectively, suggesting differential regulation between petal lobe regions. Although petal size was smaller in compacta ?hnlich than in wild type, conical cells were significantly bigger, suggesting a compensation mechanism involved in petal development. Grandiflora had larger cells in petals and increased cell division in stamens and styles, suggesting a relationship between genes controlling organ size and organ identity. The level of ploidy in petals of Grandiflora and coan was found to be equivalent to wild type petals and leaves, ruling out an excess of growth via endoreduplication. We discuss our results in terms of current models about control of lateral organ size.