Developmental characterization of the fasciated locus and mapping of Arabidopsis candidate genes involved in the control of floral meristem size and carpel number in tomato.

Mutation at the fasciated locus was a key step in the production of extreme fruit size during tomato domestication. To shed light on the nature of these changes, near-isogenic lines were used for a comparative developmental study of fasciated and wild-type tomato plants. The fasciated gene directly affects floral meristem size and is expressed before the earliest stages of flower organogenesis. As a result, mature fruit of fasciated mutants have more carpels (locules) and greater fruit diameter and mass. The discovery that fasciated affects floral meristem size led to a search for candidate genes from Arabidopsis known to be involved in floral meristem development. Putative homologs were identified in a large tomato EST database, verified through phylogenetic analyses, and mapped in tomato; none mapped to the fasciated locus; however, putative homologs of WUS and WIG mapped to the locule number locus on chromosome 2, the second major transition to large tomato fruit, with WUS showing the highest association. In other cases, minor QTLs for floral organ number (lcn2.2) and (stn11.2) co-localized with a CLV1 paralog and with the syntenic region containing the CLV3 gene in Arabidopsis, respectively.     

Effect of lateral suppressor on petal initiation in tomato

Flowers developing on tomato ( Lycopersicon esculentum ) plants homozygous for the lateral suppressor ( ls ) mutation lack petals. Scanning electron micrographs revealed that in ls plants no second whorl organs were initiated. The initiation of first, third, and fourth whorl organs were unaffected by this mutation. To investigate interactions between the cells in different layers of the floral meristem during organ initiation, a periclinal chimera between wild-type and ls tomato was generated. Flowers of the chimera having ls cells in the outer meristem layer (L1) and wild-type cells in internal layers (L2 and L3) developed normally, including the initiation of organ primordia that differentiated as petals in normal positions within the second whorl. L1 of the chimera developed in a non-autonomous manner during petal development. Thus, wild-type cells occupying the internal meristem layers provided developmental cues necessary for initiation of petal primordia at appropriate positions on the floral meristem. L1 cells carrying the lateral suppressor mutation were fully capable of responding to this information and differentiated appropriately.     

Activation of Basal Cells of the Apical Meristem during Sepal Formation in Tomato

Although great advances have been made in research on the regulation of primordium fate in the floral meristem, our understanding of the molecular events occurring during the floral transition remains incomplete. Via a careful analysis of the expression patterns of five genes encoding housekeeping functions during the floral transition in tomato (using both in situ hybridization and enzyme histochemistry), we identified a particular phase of floral development (sepal initiation) at which cells located toward the base of the meristem show a high level of cellular metabolism, whereas cells at the tip of the meristem dome show little activity. At other stages of floral development, the probes used showed genespecific patterns of expression generally consistent with our previous investigation of the vegetative apical meristem. Our data, in conjunction with other reports in the literature, enabled us to postulate that relative activation of basal cells of the meristem may be of general occurrence during the transition to flowering. Such a hypothesis could account for recent observations using periclinal chimeras on the effect of L3 genotypes on flower development and have a bearing on the expected mechanism by which the number of primordia generated by a floral meristem is regulated.     

FLORAL ONTOGENY OF ILLICIUM FLORIDANUM,WITH EMPHASIS ON STAMEN AND CARPEL DEVELOPMENT

The ontogeny of the flower and fruit of Illicium floridanum Ellis, the Star Anise, was investigated. Each of 5 or 6 bracts in each mixed terminal bud subtends either a vegetative or floral bud. The solitary flowers occur in terminal or axillary positions. Each flower has 3–6 subtending bracteoles arranged in a clockwise helix. The flowers in our material have 24–28 tepals, 30–39 stamens, and usually 13 (rarely 19) uniovulate carpels. Tepals and stamens are initiated in a low-pitched helix; carpels later appear whorled, but arise successively at different levels on the apical flanks. The floral apex is high-convex in outline with a tunica-corpus configuration; it increases in height and width throughout initiation of the floral appendages. Tepals, stamens, and carpels are initiated by one to several periclinal divisions in the subsurface layers low on the apical flanks, augmented by cell divisions in the outer layers of the corpus. The carpel develops as a conduplicate structure with appressed, connivent margins. Procambium development of floral appendages is acropetal and continuous. Bracteoles, tepals, stamens and carpels are each supplied by 1 trace; the carpellary trace splits into a dorsal and an ascending ventral sympodium. The latter bifurcates to form 2 ventral bundles. The ovular bundle diverges from the ventral sympodium. Ovule initiation occurs in a median axillary position to the carpel, an unusual type of ovule initiation. The fruit vasculature is greatly amplified as the receptacle and follicles enlarge. After carpel initiation an apical residuum persists which is not vascularized; a plate meristem develops over its surface to produce a papillate structure.     

HISTOGENESIS OF THE ANDROECIUM AND GYNOECIUM IN DOWNINGIA BACIGALUPII

A histogenetic investigation of the synandrous androecium and syncarpous gynoecium in the flower of Downingia bacigalupii Weiler (Campanulaceae; Lobelioideae) was undertaken for the purpose of comparing the modes of initiation, early growth and fusion in these floral whorls with that reported previously for the perianth in this species. Stamens are initiated as separate organs from the second tunica layer and underlying corpus regions of the concave floral meristem. Subsequent growth of stamens involves apical and intercalary growth in length and rudimentary marginal growth in breadth. Tissues of the four microsporangia originate from hypodermal sporangial initial cells and the filament is formed by intercalary growth at the base of the anther. Lateral fusion of stamens is ontogenetic and involves cuticular fusion of adjacent epidermal layers. The two emergent carpel primordia arise as crescentic organs by periclinal divisions in the second tunica layer and corpus zones. Carpel primordia also undergo apical and intercalary growth in length as well as extensive marginal growth in breadth. Radial growth in carpels is mediated by an adaxial meristem which shows its greatest concentration of activity at the carpel margins. Carpel fusion appears to be partially ontogenetic accompanied by zonal growth. Closure of the stylar canal is by the formation of a transmitting tissue derived from the protodermal layers of the adaxial carpel surfaces. A discoid nectary is initiated around the base of the style and formation of the inferior ovary is by intercalary growth of the base of the concave floral bud. The two parietal placentae originate as longitudinal outgrowths from the walls of the floral cup. Ovule initiation is simultaneous at first and then intercalary during subsequent elongation of the ovary. The ovules are anatropous, unitegmic and tenuinucellate. Stamen and carpel procambium shows a slight delay in differentiation when compared to that reported for the perianth and bract, but in all other respects carpels resemble other floral organs in their patterns of histogenesis and early growth. Stamens diverge from the other floral organs in their early pattern of growth, but a consideration of all features of their histogenesis suggests an appendicular rather than an axial interpretation of these organs.     

Defective cell proliferation in the floral meristem of alloplasmic plants of Nicotiana tabacum leads to abnormal floral organ development and male sterility

Flowers of an alloplasmic male-sterile tobacco line, comprised of the nuclear genome of Nicotiana tabacum and the cytoplasm of Nicotiana repanda, develop short, poorly-pigmented petals and abnormal sterile stamens that often are fused with the carpel wall. The development of flower organ primordia and establishment of boundaries between the different zones in the floral meristem were investigated by performing expression analysis of the tobacco orthologs of the organ identity genes GLO, AG and DEF. These studies support the conclusion that boundary formation was impaired between the organs produced in whorls 3 and 4 resulting in partial fusions between anthers and carpels. According to the investigations cell divisions and floral meristem size in the alloplasmic line were drastically reduced in comparison with the male-fertile tobacco line. The reduction in cell divisions leads to a discrepancy between cell number and cell determination at the stage when petal and stamen primordia should be initiated. At the same stage expression of the homeotic genes was delayed in comparison with the male-fertile line. However, the abnormal organ development was not due to a failure in the spatial expression of the organ identity genes. Instead the aberrant development in the floral organs of whorls 2, 3 and 4 appears to be caused by deficient floral meristem development at an earlier stage. Furthermore, defects in cell proliferation in the floral meristem of the alloplasmic male-sterile line correlates with presence of morphologically modified mitochondria. The putative causes of reduced cell number in the floral meristem and the consequences for floral development are discussed.     

Floral development of dioecious species and trends of floral evolution in Piper sensu lato

The initiation of the floral parts (mainly stamens and carpels) is described for the four dioecious species of Piper: Piper polysyphorum C. DC, P. bavinum C. DC., P. pedicellatum C. DC., P. pubicatulum C. DC. The initiation order resembles that in the perfect flowers of some species, such as P. amalago. The carpels are initiated simultaneously, in most cases, as three primordia. In P. polysyphorum , carpel tips split into two lobes, so that finally a four- or five-lobed stigma will be formed when the ovary is fully developed. The staminodes (exactly, staminodial primordia) in the female flowers are initiated in the same order as the stamens in the male flowers and remain until the ovaries are enclosed. The unisexual flowers have stamens reduced to three or two. The reduction of stamen or staminode (staminodial primordium) number is accompanied by the change of their positions from opposite the carpels to alternate. After the initiation of the staminodes, or, exactly staminodial primordia, in the female flowers, the central part of the floral apex forms a ring meristem which is triangular. The carpel primordia (often three) are initiated on the three points of the ring meristem. The evolutionary trends of the flowers of Piper sensu lato are discussed.     

Interactions between jointless and wild-type tomato tissues during development of the pedicel abscission zone and the inflorescence meristem.

The jointless mutation of tomato results in the formation of flower pedicels that lack an abscission zone and inflorescence meristems that revert to vegetative growth. We have analyzed periclinal chimeras and mericlinal sectors of jointless and wild-type tissue to determine how cells in different meristem layers (L1, L2, and L3) and their derivatives interact during these two developmental processes. Cells in the inner meristem layer, L3, alone determined whether the meristem maintained the inflorescence state or reverted to vegetative growth. Moreover, L3 derivatives determined whether a functional pedicel abscission zone formed. Limited and disorganized autonomous development of wild-type L2-derived cells occurred when they overlay mutant tissue. Adjacent mutant and wild-type L3-derived tissues in pedicels developed autonomously, indicating little or no lateral communication. Only the outermost L3-derived cells within the pedicel were capable of orchestrating normal pedicel development in overlying tissues, revealing the special status of those cells as coordinators of development for L1- and L2-derived cells, whereas the innermost L3-derived cells developed autonomously but did not influence the development of other cells.     

Cell fate in the development of the Arabidopsis flower

The Arabidopsis flower consists of four concentric whorls of organs. The first (outermost) whorl consists of four sepals and the fourth (innermost) whorl is made up of two carpels. Cell fate in the first and fourth whorls was studied using X-ray-induced yellow ch-42 sectors. Sector boundaries were found to be non-random around the two whorls and four generalizations relating the marked and unmarked tissues were deduced. In the sepal and carpel whorls the smallest sectors of marked and unmarked tissue were found to be one half of a sepal and one half of a carpel, respectively. A detailed frequency-distance map of the floral primordium was made and found to be a ridge with the fourth whorl carpels at the summit and the first whorl transverse sepal pair at the base. Consideration of: the rate of loss of chimerism in the inflorescence meristem, the frequency-distance across the flower and the frequency-distance between successive flowers, was used to produce an abstract model of the inflorescence meristem.     

Simulation of organ patterning on the floral meristem using a polar auxin transport model

An intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the phyllotaxis of leaf formation in a spiral fashion, a model study of the role of auxin transport in whorled organ patterning in the expanding floral meristem is not available yet. We present an initial simulation approach to study the mechanisms that are expected to play an important role. Starting point is a confocal imaging study of Arabidopsis floral meristems at consecutive time points during flower development. These images reveal auxin accumulation patterns at the positions of the organs, which strongly suggests that the role of auxin in the floral meristem is similar to the role it plays in the shoot apical meristem. This is the basis for a simulation study of auxin transport through a growing floral meristem, which may answer the question whether auxin transport can in itself be responsible for the typical whorled floral pattern. We combined a cellular growth model for the meristem with a polar auxin transport model. The model predicts that sepals are initiated by auxin maxima arising early during meristem outgrowth. These form a pre-pattern relative to which a series of smaller auxin maxima are positioned, which partially overlap with the anlagen of petals, stamens, and carpels. We adjusted the model parameters corresponding to properties of floral mutants and found that the model predictions agree with the observed mutant patterns. The predicted timing of the primordia outgrowth and the timing and positioning of the sepal primordia show remarkable similarities with a developing flower in nature.     

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.     

The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa

In this article, we report that carpel specification in the Oryza sativa (rice) flower is regulated by the floral homeotic gene DROOPING LEAF (DL) that is distinct from the well-known ABC genes. Severe loss-of-function mutations of DL cause complete homeotic transformation of carpels into stamens. Molecular cloning reveals that DL is a member of the YABBY gene family and is closely related to the CRABS CLAW (CRC) gene of Arabidopsis thaliana. DL is expressed in the presumptive region (carpel anlagen), where carpel primordia would initiate, and in carpel primordia. These results suggest that carpel specification is regulated by DL in rice flower development. Whereas CRC plays only a partial role in carpel identity, DL may have been recruited to have the more essential function of specifying carpels during the evolution of rice. We also show that DL interacts antagonistically with class B genes and controls floral meristem determinacy. In addition, severe and weak dl alleles fail to form a midrib in the leaf. The phenotypic analysis of dl mutants, together with analyses of the spatial expression patterns and ectopic expression of DL, demonstrate that DL regulates midrib formation by promoting cell proliferation in the central region of the rice leaf.     

Regulation of SUP expression identifies multiple regulators involved in arabidopsis floral meristem development

During the course of flower development, floral homeotic genes are expressed in defined concentric regions of floral meristems called whorls. The SUPERMAN (SUP, also called FLO10) gene, which encodes a C2H2-type zinc finger protein, is involved in maintenance of the stamen/carpel whorl boundary (the boundary between whorl 3 and whorl 4) in Arabidopsis. Here, we show that the regulation of SUP expression in floral meristems is complex, consisting of two distinct phases, initiation and maintenance. The floral meristem identity gene LEAFY (LFY) plays a role in the initiation phase through at least two pathways, which differ from each other in the involvement of two homeotic genes, APETALA3 (AP3) and PISTILLATA (PI). AP3, PI, and another homeotic gene, AGAMOUS (AG), are further required for SUP expression in the later maintenance phase. Aside from these genes, there are other as yet unidentified genes that control both the temporal and spatial patterns of SUP expression in whorl 3 floral meristems. SUP appears to act transiently, probably functioning to trigger a genetic circuit that creates the correct position of the whorl 3/whorl 4 boundary.     

Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants.

To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.     

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.     

ORGANOGENESIS IN THE CARPELLATE FLOWER OF DRIMYS LANCEOLATA

This study of floral development in Drimys lanceolata in Section Tasmannia provides a basis for comparison with D. winteri, a member of the section Wintera, which has been described previously. The carpellate flowers of D. lanceolata have 2 sepals, 4–6 petals, and a solitary carpel, which form in acropetal succession. In symmetry the flower and its apical meristem are bilateral rather than radial, as in the flower of Drimys winteri. The floral apex of D. lanceolata is zonate while that of D. winteri is organized as a mantle and core. Preceding carpel initiation the floral apex of D. lanceolata is narrowly wedge-shaped, while that of D. winteri is low-convex. The entire apex is utilized in carpel initiation in D. lanceolata, involving many subsurface cell divisions over the entire summit. No apical residuum remains, and the carpel is terminal. In this feature the contrast with D. winteri is particularly marked, since in the latter, carpels are initiated laterally around the floral apex, which c an be recognized as an apical residuum after all appendages have formed.