Floral Organ Specification Research Articles

Modelling the Molecular Interactions in the Flower Developmental Network of Arabidopsis thaliana

We present a dynamical model of the gene network controlling flower development in Arabidopsis thaliana. The network is centered at the regulation of the floral organ identity genes (AP1, AP2, AP3, PI and AG) and ends with the transcription factor complexes responsible for differentiation of floral organs. We built and simulated the regulatory interactions that determine organ specificity using an extension of hybrid Petri nets as implemented in Cell Illustrator. The network topology is characterized by two main features: (1) the presence of multiple autoregulatory feedback loops requiring the formation of protein complexes, and (2) the role of spatial regulators determining floral patterning. The resulting network shows biologically coherent expression patterns for the involved genes, and simulated mutants produce experimentally validated changes in organ expression patterns. The requirement of heteromeric higher-order protein complex formation for positive autoregulatory feedback loops attenuates stochastic fluctuations in gene expression, enabling robust organ-specific gene expression patterns. If autoregulation is mediated by monomers or homodimers of proteins, small variations in initial protein levels can lead to biased production of homeotic proteins, ultimately resulting in homeosis. We also suggest regulatory feedback loops involving miRNA loci by which homeotic genes control the activity of their spatial regulators.

The homeotic gene long sterile lemma ( G1 ) specifies sterile lemma identity in the rice spikelet

The mechanism of floral organ specification is principally conserved in angiosperms, as demonstrated by the ABC model. By contrast, mechanisms that regulate the development of organs or structures specific to a group of species remain unclear. Grasses have unique inflorescence units, comprising spikelets and florets. In the genus Oryza (rice), the single spikelet consists of a fertile floret subtended by a lemma and a palea, two sterile lemmas, and rudimentary glumes. Each sterile lemma is a tiny glume-like organ with a smooth surface. Here, we have examined a long sterile lemma1 (g1) mutant, in which the sterile lemma is enlarged like the lemma. Detailed phenotypic analysis reveals that the large sterile lemma in the g1 mutant appears to be caused by homeotic transformation of the sterile lemma into a lemma, suggesting that G1 is involved in the repression of lemma identity to specify the sterile lemma. Gene isolation reveals that G1 is a member of a plant-specific gene family that encodes proteins with a previously uncharacterized domain, named here ALOG (Arabidopsis LSH1 and Oryza G1). G1 mRNA is expressed in sterile lemma primordia throughout their development, and G1 protein is localized in the nucleus. A trans-activation assay using the yeast GAL4 system suggests that G1 is involved in transcriptional regulation. Repression of lemma identity by G1 is consistent with a hypothesis proposed to explain the morphological evolution of rice spikelets. We also show that a wild rice species, Oryza grandiglumis, that forms large sterile lemmas has serious mutations in the G1 gene.

RA68 is required for postmeiotic pollen development in Oryza sativa

Postmeiotic development is a unique characteristic of flowering plants. During the development, microspores undergo two cycles of mitosis (PMI and PMII) and a subsequent maturation process to finally produce the mature pollen, but the mechanism underlying the development is still largely unknown. Here, we report on the roles of a novel gene, RA68, in postmeiotic pollen development in Oryza sativa. RA68 was expressed preferentially in shoots and flowers. In flowers, the transcript persisted from the floral organ differentiation to the mature pollen stages and showed preferential accumulation in male meiocytes, developing pollen and tapetal cells. RA68-deficient RNAi lines showed reduced seed setting and pollen viability but not an aberrant phenotype in vegetative organs. Knockdown of RA68 led to arrested PMI, smaller pollen grains with little or no starch, and aborted pollen but not severely distruped male meiosis. Additionally, no abnormality of anther wall development was observed in RA68-RNAi lines. RA68 may be required for postmeiotic pollen development by affecting PMI and starch accumulation.

Gibberellin as a factor in floral regulatory networks

Gibberellins (GAs) function not only to promote the growth of plant organs, but also to induce phase transitions during development. Their involvement in flower initiation in long-day (LD) and biennial plants is well established and there is growing insight into the mechanisms by which floral induction is achieved. The extent to which GAs mediate the photoperiodic stimulus to flowering in LD plants is, with a few exceptions, less clear. Despite evidence for photoperiod-enhanced GA biosynthesis in leaves of many LD plants, through up-regulation of GA 20-oxidase gene expression, a function for GAs as transmitted signals from leaves to apices in response to LD has been demonstrated only in Lolium species. In Arabidopsis thaliana, as one of four quantitative floral pathways, GA signalling has a relatively minor influence on flowering time in LD, while in SD, in the absence of the photoperiod flowering pathway, the GA pathway assumes a major role and becomes obligatory. Gibberellins promote flowering in Arabidopsis through the activation of genes encoding the floral integrators SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), LEAFY (LFY), and FLOWERING LOCUS T (FT) in the inflorescence and floral meristems, and in leaves, respectively. Although GA signalling is not required for floral organ specification, it is essential for the normal growth and development of these organs. The sites of GA production and action within flowers, and the signalling pathways involved are beginning to be revealed.

Floral Morphogenesis: Stochastic Explorations of a Gene Network Epigenetic Landscape

In contrast to the classical view of development as a preprogrammed and deterministic process, recent studies have demonstrated that stochastic perturbations of highly non-linear systems may underlie the emergence and stability of biological patterns. Herein, we address the question of whether noise contributes to the generation of the stereotypical temporal pattern in gene expression during flower development. We modeled the regulatory network of organ identity genes in the Arabidopsis thaliana flower as a stochastic system. This network has previously been shown to converge to ten fixed-point attractors, each with gene expression arrays that characterize inflorescence cells and primordial cells of sepals, petals, stamens, and carpels. The network used is binary, and the logical rules that govern its dynamics are grounded in experimental evidence. We introduced different levels of uncertainty in the updating rules of the network. Interestingly, for a level of noise of around 0.5–10%, the system exhibited a sequence of transitions among attractors that mimics the sequence of gene activation configurations observed in real flowers. We also implemented the gene regulatory network as a continuous system using the Glass model of differential equations, that can be considered as a first approximation of kinetic-reaction equations, but which are not necessarily equivalent to the Boolean model. Interestingly, the Glass dynamics recover a temporal sequence of attractors, that is qualitatively similar, although not identical, to that obtained using the Boolean model. Thus, time ordering in the emergence of cell-fate patterns is not an artifact of synchronous updating in the Boolean model. Therefore, our model provides a novel explanation for the emergence and robustness of the ubiquitous temporal pattern of floral organ specification. It also constitutes a new approach to understanding morphogenesis, providing predictions on the population dynamics of cells with different genetic configurations during development.

Hierarchical Developmental Morphology: The Case of the Inflorescence ofPhilodendron ornatum(Araceae)

This study deals specifically with floral organogenesis and the development of the inflorescence of Philodendron ornatum. The development of the inflorescence of P. ornatum can be divided into two morphogenetic phases: (i) before and (ii) after the initiation of the floral organs. Before the initiation of the floral organs, the ratio between the width and the length of the inflorescence is greater than after their initiation. The first phase corresponds to the establishment of the phyllotactic pattern, and the second phase corresponds to the differentiation of floral organs. Pistillate flowers are initiated on the lower portion of the inflorescence, and the staminate flowers are initiated on the distal portion. An intermediate zone consisting of sterile male flowers and atypical bisexual flowers (ABFs) with carpels and staminodes inserted on the same whorl is also present. The portion of the ABFs facing the male zone forms staminodes, and the portion facing the female zone develops an incomplete gynoecium with a few carpels. This phenomenon raises the issue of relationships between hierarchical levels and their mutual influence on the developmental morphology of ABFs. In this article, this question is addressed in the context of the basic triadic system developed by Salthe.

Type specification and spatial pattern formation of floral organs: A dynamic development model

A mathematical model simulating spatial pattern formation (positioning) of floral organs is proposed. Computer experiment with this model demonstrated the following sequence of spatial pattern formation in a typical cruciferous flower: medial sepals, carpels, lateral sepals, long stamens, petals, and short stamens. The positioning was acropetal for the perianth organs and basipetal for the stamens and carpels. Organ type specification and positioning proceed non-simultaneously in different floral parts and organ type specification goes ahead of organ spatial pattern formation. Computer simulation of flower development in several mutants demonstrated that the AG and AP2 genes determine both organ type specification and formation of the zones for future organ development. The function of the AG gene is to determine the basipetal patterning zones for the development of the reproductive organs, while the AP2 gene maintains proliferative activity of the meristem establishing the acropetal patterning zone for the development of the perianth organs.

From Genes to Flower Patterns and Evolution: Dynamic Models of Gene Regulatory Networks

Genes and proteins form complex dynamical systems or gene regulatory networks (GRN) that can reach several steady states (attractors). These may be associated with distinct cell types. In plants, the ABC combinatorial model establishes the necessary gene combinations for floral organ cell specification. We have developed dynamic gene regulatory network (GRN) models to understand how the combinatorial selection of gene activity is established during floral organ primordia specification as a result of the concerted action of ABC and non-ABC genes. Our analyses have shown that the floral organ specification GRN reaches six attractors with gene configurations observed in primordial cell types during early stages of flower development and four that correspond to regions of the inflorescence meristem. This suggests that it is the overall GRN dynamics rather than precise signals that underlie the ABC model. Furthermore, our analyses suggest that the steady states of the GRN are robust to random alterations of the logical functions that define the gene interactions. Here we have updated the GRN model and have systematically altered the outputs of all the logical functions and addressed in which cases the original attractors are recovered. We then reduced the original three-state GRN to a two-state (Boolean) GRN and performed the same systematic perturbation analysis. Interestingly, the Boolean GRN reaches the same number and type of attractors as reached by the three-state GRN, and it responds to perturbations in a qualitatively identical manner as the original GRN. These results suggest that a Boolean model is sufficient to capture the dynamical features of the floral network and provide additional support for the robustness of the floral GRN. These findings further support that the GRN model provides a dynamical explanation for the ABC model and that the floral GRN robustness could be behind the widespread conservation of the floral plan among eudicotyledoneous plants. Other aspects of evolution of flower organ arrangement and ABC gene expression patterns are discussed in the context of the approach proposed here.

Isolation ofmtpimProvesTnt1a Useful Reverse Genetics Tool inMedicago truncatulaand Uncovers New Aspects ofAP1-Like Functions in Legumes

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

Isolation and sequence analysis of the floral homeotic geneBAP2inBrassica rapa

AbstractTheArabidopsishomeotic geneAP2is essential for floral meristem development and organ specification. In this study, we report the isolation and sequence comparison of theAP2homologue ofBrassica rapa(BAP2). The results showed that theBAP2DNA sequence was 2138 bp in length and contained nine introns. It shared 90% identity withAP2in the coding region. The putative BAP2 polypeptide contained a nuclear localization signal (NLS) sequence and two copies of highly conserved AP2 domain, suggesting thatBAP2may function similarly toAP2. Two nucleotide variations were detected in DNA sequences from wild-type and apetalousB. rapaplants at theBAP2locus, while the putative polypeptides were identical. We propose thatBAP2is not likely to be responsible for the apetalous mutation inB. rapa.

Genome-Wide Analysis of Gene Expression during Early Arabidopsis Flower Development

Detailed information about stage-specific changes in gene expression is crucial for the understanding of the gene regulatory networks underlying development. Here, we describe the global gene expression dynamics during early flower development, a key process in the life cycle of a plant, during which floral patterning and the specification of floral organs is established. We used a novel floral induction system in Arabidopsis, which allows the isolation of a large number of synchronized floral buds, in conjunction with whole-genome microarray analysis to identify genes with differential expression at distinct stages of flower development. We found that the onset of flower formation is characterized by a massive downregulation of genes in incipient floral primordia, which is followed by a predominance of gene activation during the differentiation of floral organs. Among the genes we identified as differentially expressed in the experiment, we detected a significant enrichment of closely related members of gene families. The expression profiles of these related genes were often highly correlated, indicating similar temporal expression patterns. Moreover, we found that the majority of these genes is specifically up-regulated during certain developmental stages. Because co-expressed members of gene families in Arabidopsis frequently act in a redundant manner, these results suggest a high degree of functional redundancy during early flower development, but also that its extent may vary in a stage-specific manner.

Meristem Identity Genes and the Control of the Reproductive Development in Brassica oleracea

The genetic factors that control reproductive development in B. oleracea remain a mystery. Broccoli differs from cauliflower in its floral development stage at harvest. We are studying the role of meristem identity genes (MIGs) in the transition from inflorescence meristem (cauliflower) to floral buds (broccoli). The objectives are to determine stage-specific roles of MIGs during reproductive development and to check whether expression of flowering genes in heading B. oleracea is as predicted by the Arabidopsis flowering model. We tested a model of arrest in B. oleracea that incorporates FUL, a redundant gene of AP1 in controlling inflorescence architecture and floral meristem identity, the meristem gene TFL1, the flowering gene LFY, and AP1/CAL, and genes involved in flower transition. Conclusions. 1) Arrest at the inflorescence meristem stage is highly correlated with a decrease in LFY to TFL1 ratio, given by a decrease in TFL1 expression. 2) Transcription of AP1c is stimulated at the time of floral primordium initiation, suggesting a role in floral transition but not in floral organ specification. Plants recessive for AP1a, AP1c, and CAL formed normal floral buds containing all four whorls of organs, and did not necessarily form curd. We suggest that their ability to flower could be related with the ectopic expression of FUL by affecting TFL1 expression. FUL paralogs were highly expressed at all stages of development of the triple mutant plants. 3) The lack of upregulation in AP1 transcripts at the floral bud stage, and the absence of an A-function mutant phenotype imply that other genes act redundantly with AP1 in the specification of sepal identity and questions the role of AP1a and AP1c as A-function genes in B. oleracea.

Genome‐wide analysis of spatial and temporal gene expression in rice panicle development

The basic structure of a rice inflorescence (the panicle) is determined by the pattern of branch formation, which is established at the early stages of panicle development. In this study we conducted global transcriptome profiling of the early stages of rice panicle development from phase transition to floral organ differentiation. To generate a meristem-specific gene-expression profile, shoot apical meristems (SAMs) and subsequently formed, very young panicles were collected manually and used for cDNA microarray analysis. We identified 357 out of 22,000 genes that are expressed differentially in the early stages of panicle development, and the 357 genes were classified into seven groups based on their temporal expression patterns. The most noticeable feature is that a fairly small number of genes, which are extensively enriched in transcription factors, are upregulated in the SAM immediately after phase transition. In situ hybridization analysis showed that each gene analysed exhibits a unique and interesting localization of mRNA. Remarkably, one of the transcription factors was proven to be a close downstream component of the pathway in which LAX, a major regulator of panicle branching, acts. These results suggest that our strategy--careful collection of meristems, global transcriptome analysis and subsequent in situ hybridization analysis--is useful not only to obtain a genome-wide view of gene expression, but also to reveal genetic networks controlling rice panicle development.

Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanol-inducible alc gene-expression system

In Arabidopsis, different combinations of ABC organ identity proteins interact in the presence of SEPALLATA (SEP) proteins to regulate floral organ differentiation. Ectopic expression of SEP3 in combination with class A and B or B and C genes is sufficient to homeotically convert vegetative leaves into petal-like organs and bracts into stamen-like structures, respectively. Recently, it has been shown that the three MADS-box genes SEEDSTICK ( STK), SHATTERPROOF1 ( SHP1) and SHP2 act redundantly to control ovule identity. Protein interaction assays performed in yeast in combination with genetic studies demonstrated that these MADS-box factors only interact in the presence of SEP proteins to form complexes that determine ovule differentiation. Here, we address the question whether the ectopic co-expression of ovule identity proteins is sufficient to induce the homeotic conversion of vegetative leaves into carpel-like structures bearing ovules. We present the phenotypic characterization of Arabidopsis plants that ectopically express ovule identity factors under the regulation of the ethanol inducible gene expression system. These experiments indicate that the ectopic co-expression of SEP3 and SHP1 and/or STK is probably not sufficient to homeotically transform vegetative tissues into carpels with ovules. However, comparing the phenotypes obtained by ectopic expression of STK and/or SHP1 with or without SEP3 shows that co-expression of factors that are able to form complexes in yeast cause more extreme homeotic transformations, confirming the functional role of these complexes in vivo.

The structural and developmental characteristics of floral nectaries of Litchi chinensis and their biological significance

Floral nectaries attract insect pollinators through secreted nectar. The structure and the size of floral nectaries are determining factors of the quantity and concentration of the nectar produced, which may consequently influence the patterns of insect visitation. In this study, we examined the structural and developmental characteristics of the floral nectaries of Litchi chinensis of “Nuomici” and “Feizixiao”. In two variety of L. chinensis, the floral nectaries of both functional male and female flowers are located between the cup-shaped calyx and the ovary. These floral nectaries are typical structural nectaries, formed as protrusions on the surface of flower receptacles, and at the mature stage, they are composed of epidermis, nectariferous tissue, and vascular tissue containing only phloem elements. The epidermis of the floral nectaries is covered with numerous epidermal hairs, with sporadic stomata distributed on the top surface of the nectaries. Before the nectaries start to secret nectar, the nectariferous tissues are filled with numerous starch grains, which display regular changes before, during, and after nectar secretion. The nectariferous tissue contains two cell types of different morphological characteristics. These cell types presumably have different functions, either for nectar synthesis, or for protecting nectaries from damage by phenolic compounds. The primordia of these nectaries initiate during floral organ differentiation, and its development coincides with the formation of microspores and macrospores in the male and female flowers, respectively. Based on these structural and developmental characteristics, we discussed the ontogeny and phylogeny of the floral nectaries in L. chinensis and possible evolutional adaptation of nectaries for effective attraction of insect pollinators.

SEPALLATA gene diversification: brave new whorls

SEPALLATA (SEP) genes form an integral part of models that outline the molecular basis of floral organ determination and are hypothesized to act as co-factors with ABCD floral homeotic genes in specifying different floral whorls. The four SEP genes in Arabidopsis function redundantly, but the extent to which SEP genes in other flowering plants function similarly is unknown. Using a recent 113-gene SEP phylogeny as a framework, we find surprising heterogeneity among SEP gene C-terminal motifs, mRNA expression patterns, protein-protein interactions and inferred function. Although some SEP genes appear to function redundantly, others have novel roles in fruit maturation, floral organ specification and plant architecture, and have played a major role in floral evolution of diverse plants.

In vitro regeneration and callogenesis in tissue culture of floral organs of the genus Iris (Iridaceae)

We tested the differentiation and morphogenetic capacity of floral organs of Iris ensata, I. setosa, and I. sanguinea cultured in vitro. Organogenesis through direct formation of shoots from explants, callogenesis, and floral organogenesis were demonstrated in I. ensata callus culture in vitro. These processes depended on the plant species and on the content of phytohormones in the medium. Adventitious shoots proved to develop on the basal part of the perianth tube and on the apical part of the ovary, while roots were not formed. Direct organogenesis was induced by the following phytohormones: alpha-naphthylacetic acid and 6-benzylaminopurine for I. ensata and 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine for I. setosa and I. sanguinea; while callogenesis was induced by 2,4-dichlorophenoxyacetic acid. The obtained data indicate that development of adventitious structures from iris floral organs requires the presence of 6-benzylaminopurine in the growth medium.

Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.

Genome-Wide Analysis of Gene Expression during Early Arabidopsis Flower Development

Detailed information about stage-specific changes in gene expression is crucial for the understanding of the gene regulatory networks underlying development. Here, we describe the global gene expression dynamics during early flower development, a key process in the life cycle of a plant, during which floral patterning and the specification of floral organs is established. We used a novel floral induction system in Arabidopsis, which allows the isolation of a large number of synchronized floral buds, in conjunction with whole-genome microarray analysis to identify genes with differential expression at distinct stages of flower development. We found that the onset of flower formation is characterized by a massive downregulation of genes in incipient floral primordia, which is followed by a predominance of gene activation during the differentiation of floral organs. Among the genes we identified as differentially expressed in the experiment, we detected a significant enrichment of closely related members of gene families. The expression profiles of these related genes were often highly correlated, indicating similar temporal expression patterns. Moreover, we found that the majority of these genes is specifically up-regulated during certain developmental stages. Because co-expressed members of gene families in Arabidopsis frequently act in a redundant manner, these results suggest a high degree of functional redundancy during early flower development, but also that its extent may vary in a stage-specific manner.

Arabidopsis POLYOL TRANSPORTER5, a New Member of the Monosaccharide Transporter-Like Superfamily, Mediates H+-Symport of Numerous Substrates, Including myo-Inositol, Glycerol, and Ribose

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5-green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H+-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.