Arabidopsis Flower Development Research Articles

Roles for farnesol and ABA in Arabidopsis flower development

The Arabidopsis FOLK (At5g58560) gene encodes farnesol kinase, which phosphorylates farnesol to farnesyl phosphate. Loss-of-function mutations in the FOLK gene are associated with enhanced sensitivity to abscisic acid (ABA), suggesting that FOLK negatively regulates ABA signaling. Moreover, folk flowers develop supernumerary carpels under water stress, providing evidence for a molecular link between farnesol metabolism, abiotic stress signaling and flower development. Here, we show that farnesol increases ABA sensitivity and that ABA affects flower development in Arabidopsis.

Aintegumenta and Aintegumenta-Like6 regulate auxin-mediated flower development in Arabidopsis

BackgroundTwo related genes encoding AP2/ERF-type transcription factors, AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6), are important regulators of floral growth and patterning in Arabidopsis. Evidence suggests that these genes promote several aspects of flower development in response to auxin. To investigate the interplay of ANT, AIL6 and auxin during floral development, I have examined the phenotypic consequences of disrupting polar auxin transport in ant, ail6 and ant ail6 mutants by either genetic or chemical means.ResultsPlants containing mutations in ANT or AIL6 alone or in both genes together exhibit increased sensitivity to disruptions in polar auxin transport. Both genes promote shoot growth, floral meristem initiation and floral meristem patterning in combination with auxin transport. However, differences in the responses of ant and ail6 single mutants to perturbations in auxin transport suggest that these two genes also have non-overlapping activities in each of these developmental processes.ConclusionsThe enhanced sensitivity of ant and ail6 mutants to alterations in polar auxin transport suggests that these mutants have defects in some aspect of auxin physiology. The inability of ant ail6 double mutants to initiate flowers in backgrounds disrupted for auxin transport confirm the proposed roles for these two genes in floral meristem initiation.

Auxin regulation of Arabidopsis flower development involves members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) family

Auxin is an important regulator of many aspects of plant growth and development. During reproductive development, auxin specifies the site of flower initiation and subsequently regulates organ growth and patterning as well as later events that determine reproductive success. Underlying auxin action in plant tissues is its uneven distribution, resulting in groups of cells with high auxin levels (auxin maxima) or graded distributions of the hormone (auxin gradients). Dynamic auxin distribution within the periphery of the inflorescence meristems specifies the site of floral meristem initiation, while auxin maxima present at the tips of developing floral organ primordia probably mediate organ growth and patterning. The molecular means by which auxin accumulation patterns are converted into developmental outputs in flowers is not well understood. Members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family are important developmental regulators in both roots and shoots. In roots, the expression of two AIL/PLT genes is regulated by auxin and these genes feed back to regulate auxin distribution. Here, several aspects of flower development involving both auxin and AIL/PLT activity are described, and evidence linking AIL/PLT function with auxin distribution in reproductive tissues is presented.

Farnesol kinase is involved in farnesol metabolism, ABA signaling and flower development in Arabidopsis

Farnesol, which is toxic to plant cells at high concentrations, is sequentially phosphorylated to farnesyl phosphate and farnesyl diphosphate. However, the genes responsible for the sequential phosphorylation of farnesol have not been identified and the physiological role of farnesol phosphorylation has not been fully elucidated. To address these questions, we confirmed the presence of farnesol kinase activity in Arabidopsis (Arabidopsis thaliana) membranes and identified the corresponding gene (At5g58560, FOLK). Heterologous expression in recombinant yeast cells established farnesol as the preferred substrate of the FOLK-encoded kinase. Moreover, loss-of-function mutations in the FOLK gene abolished farnesol kinase activity, caused an abscisic acid-hypersensitive phenotype and promoted the development of supernumerary carpels under water-stress conditions. In wild-type plants, exogenous abscisic acid repressed FOLK gene expression. These observations demonstrate a role for farnesol kinase in negative regulation of abscisic acid signaling, and provide molecular evidence for a link between farnesol metabolism, abiotic stress signaling and flower development.

PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants

A MADS-box gene was isolated using the suppressive subtractive hybridization library between early-flowering mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.). This gene is highly homologous with Arabidopsis SHORT VEGETATIVE PHASE (SVP). Based on real-time PCR and in situ hybridization during bud differentiation, PtSVP was expressed intensively in dormant tissue and vegetative meristems. PtSVP transcripts were detected in apical meristems before floral transition, then down-regulated during the transition. PtSVP expression was higher in differentiated (flower primordium) than in undifferentiated cells (apical meristems). The PtSVP expression pattern during apical meristem determination suggested that its function is not to depress flower initiation but to maintain meristem development. Transcription of PtSVP in Arabidopsis svp-41 showed partially rescued SVP function. Ectopic overexpression of PtSVP in wild-type Arabidopsis induced late flowering similar to the phenotypes induced by other SVP/StMADS-11-like genes, but transformants produced additional trichomes and floral defects, such as flower-like structures instead of carpels. Ectopic expression of PtSVP in tobacco also caused additional florets. Overexpression of PtSVP in tobacco inhibited early transition of the coflorescence and prolonged coflorescence development, thus causing additional florets at the later stage. A yeast two-hybrid assay indicated that PtSVP significantly interacted with PtAP1, a homolog of Arabidopsis APETALA1 (AP1). These findings suggest that citrus SVP homolog genes are involved in flowering time regulation and may influence inflorescence meristem identity in some conditions or genetic backgrounds. SVP homologs might have evolved among plant species, but the protein functions are conserved between Arabidopsis and citrus.

BROTHER OF FT AND TFL1 (BFT) has TFL1-like activity and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis.

The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family that encodes important regulators that control flower development in Arabidopsis. Here, we investigated the biological role of the product of BROTHER OF FT AND TFL1 (BFT), a member of this family, whose function remains unknown. Comparison of the critical residues that play a role in distinguishing FT- or TFL1-like activity revealed that BFT is more similar to FT. Similar to FT expression, BFT expression showed a diurnal oscillation pattern, peaking in the evening. In situ hybridization revealed BFT expression in the shoot apical meristem, young leaf and axillary inflorescence meristem. Transgenic plants over-expressing BFT exhibited delayed flowering and severe floral defects (floral indeterminacy and compact inflorescences surrounded by serrate leaves), similar to 35S::TFL1 plants. LEAFY (LFY) and APETALA1 (AP1) expression was significantly reduced in 35S::BFT plants. BFT over-expression failed to rescue the terminal flower phenotype of tfl1 mutants; however, it delayed both terminal flower formation in the primary inflorescence and axillary inflorescence development in the tfl1 mutant background. Consistent with this, the loss-of-function BFT alleles, bft-2 and an BFT RNAi line, accelerated termination of the primary inflorescence and formation of axillary inflorescences in the tfl1 mutant background. Taken together, our results suggest that, despite similarities in the critical residues of BFT and FT, BFT possesses a TFL1-like activity and functions redundantly with TFL1 in inflorescence meristem development, and possibly contributes to the regulation of plant architecture.

A B Functional Gene Cloned from Lily Encodes an Ortholog of Arabidopsis PISTILLATA (PI)

The classical ABC model proposed for flower development in Arabidopsis and Antirrhinum appropriately sheds light on the biological process of flower development and differentiation and serves in manipulating the floral structure of other important ornamental plants. In this study, LLGLO1, a B functional gene from Lilium longiflorum was isolated and characterized. RT-PCR analysis elucidated that temporal and spatial expression pattern of LLGLO1. This putative gene was strongly expressed in 1, 2, and 3 whorl organs, i.e., outer whorl tepals, inner whorl tepals, and stamens. Genetic effect of LLGLO1 was assayed by ectopic expression in model plant Arabidopsis. Transformed plants showed homeotic transformation of sepals into petaloid sepals in the first whorl, which is similar to the transgenic plants of 35S::PI. So LLGLO1 was one member of GLO/PI sub-family gene to function in flower development.

Floral stem cells: from dynamic balance towards termination

During early flower development in Arabidopsis, floral stem cells proliferate and produce a sufficient amount of cells that are recruited for organogenesis. However, after the central organ primordia initiate, stem cell activity in the floral meristem is terminated to ensure the differentiation of a fixed number of floral organs. Underlying this process, the genetic programme regulating the fate of floral meristems undergoes a shift from a spatially balanced signalling scheme for stem cell maintenance to a temporally controlled transcriptional scheme for stem cell termination. Precise timing of stem cell termination is a key issue for flower development, which is secured by the orchestration of multiple regulators in transcriptional and epigenetic regulation.

The flowering of Arabidopsis flower development

Flowers come in a variety of colors, shapes and sizes. Despite this variety, flowers have a very stereotypical architecture, consisting of a series of sterile organs surrounding the reproductive structures. Arabidopsis, as the premier model system for molecular and genetic analyses of plant development, has provided a wealth of insights into how this architecture is specified. With the advent of the completion of the Arabidopsis genome sequence a decade ago, in combination with a rich variety of forward and reverse genetic strategies, many of the genes and regulatory pathways controlling flower initiation, patterning, growth and differentiation have been characterized. A central theme that has emerged from these studies is the complexity and abundance of both positive and negative feedback loops that operate to regulate different aspects of flower formation. Presumably, this considerable degree of feedback regulation serves to promote a robust and stable transition to flowering, even in the face of genetic or environmental perturbations. This review will summarize recent advances in defining the genes, the regulatory pathways, and their interactions, that underpin how the Arabidopsis flower is formed.

Cell‐type specific analysis of translating RNAs in developing flowers reveals new levels of control

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.

Flower development.

Flowers are the most complex structures of plants. Studies of Arabidopsis thaliana, which has typical eudicot flowers, have been fundamental in advancing the structural and molecular understanding of flower development. The main processes and stages of Arabidopsis flower development are summarized to provide a framework in which to interpret the detailed molecular genetic studies of genes assigned functions during flower development and is extended to recent genomics studies uncovering the key regulatory modules involved. Computational models have been used to study the concerted action and dynamics of the gene regulatory module that underlies patterning of the Arabidopsis inflorescence meristem and specification of the primordial cell types during early stages of flower development. This includes the gene combinations that specify sepal, petal, stamen and carpel identity, and genes that interact with them. As a dynamic gene regulatory network this module has been shown to converge to stable multigenic profiles that depend upon the overall network topology and are thus robust, which can explain the canalization of flower organ determination and the overall conservation of the basic flower plan among eudicots. Comparative and evolutionary approaches derived from Arabidopsis studies pave the way to studying the molecular basis of diverse floral morphologies.

Analysis of the Arabidopsis Floral Proteome: Detection of over 2 000 Proteins and Evidence for Posttranslational Modifications

The proteome of the Arabidopsis flower has not been extensively studied previously. Here, we report a proteomic analysis of the wild type Arabidopsis flower. Using both two-dimensional electrophoresis/mass spectrometry (2-DGE/MS) and multi-dimensional protein identification technology (MudPIT) approaches, we identified 2,446 proteins. Although a single experiment or analysis uncovered only a subset of the proteins we identified, a combination of multiple experiments and analyses facilitated the detection of a greater number of proteins. When proteins are grouped according to RNA expression levels revealed by microarray experiments, we found that proteins encoded by genes with relatively high levels of expression were detected with greater frequencies. On the other hand, at the level of the individual gene/protein, there was not a good correlation between protein spot intensity and microarray values. We also obtained strong evidence for post-translational modification from 2-DGE and MudPIT data. We detected proteins that are annotated to function in protein synthesis, folding, modification, and degradation, as well as the presence of regulatory proteins such as transcription factors and protein kinases. Finally, sequence and evolutionary analysis of genes for active methyl group metabolisms suggests that these genes are highly conserved. Our results allow the formulation of hypotheses regarding post-translational regulation of proteins in the flower, providing new understanding about Arabidopsis flower development and physiology.

Possible targets of “magic spots” in plant signalling

The prokaryotic signalling molecules (p)ppGpp, also called “magic spots”, regulate a wide variety of physiological activities in bacteria, including transcription, translation, and replication as well as some enzymatic activities such as those of some GTP-binding proteins, which are necessary for bacterial cells to adapt their physiology to different environmental stimuli. This response is called the stringent response. Recently, (p)ppGpp molecules and (p)ppGpp synthetase homologues, designated RSHs, have been identified in plants. At least some of the RSHs are targeted to chloroplasts. A knockdown mutation in one of the RSHs results in unusual flower development in Arabidopsis, suggesting that the plastid stringent response has important roles in the physiology of higher plants. Possible (p)ppGpp target proteins are investigated.Addendum to: Mizusawa K, Masuda S, Ohta H. Expression profiling of four RelA/SpoT-like proteins, homologues of bacterial stringent factors, in Arabidopsis thaliana. Planta 2008; 228:553-62.

Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis.

BackgroundThe Wuschel related homeobox (WOX) family proteins are key regulators implicated in the determination of cell fate in plants by preventing cell differentiation. A recent WOX phylogeny, based on WOX homeodomains, showed that all of the Physcomitrella patens and Selaginella moellendorffii WOX proteins clustered into a single orthologous group. We hypothesized that members of this group might preferentially share a significant part of their function in phylogenetically distant organisms. Hence, we first validated the limits of the WOX13 orthologous group (WOX13 OG) using the occurrence of other clade specific signatures and conserved intron insertion sites. Secondly, a functional analysis using expression data and mutants was undertaken.ResultsThe WOX13 OG contained the most conserved plant WOX proteins including the only WOX detected in the highly proliferating basal unicellular and photosynthetic organism Ostreococcus tauri. A large expansion of the WOX family was observed after the separation of mosses from other land plants and before monocots and dicots have arisen. In Arabidopsis thaliana, AtWOX13 was dynamically expressed during primary and lateral root initiation and development, in gynoecium and during embryo development. AtWOX13 appeared to affect the floral transition. An intriguing clade, represented by the functional AtWOX14 gene inside the WOX13 OG, was only found in the Brassicaceae. Compared to AtWOX13, the gene expression profile of AtWOX14 was restricted to the early stages of lateral root formation and specific to developing anthers. A mutational insertion upstream of the AtWOX14 homeodomain sequence led to abnormal root development, a delay in the floral transition and premature anther differentiation.ConclusionOur data provide evidence in favor of the WOX13 OG as the clade containing the most conserved WOX genes and established a functional link to organ initiation and development in Arabidopsis, most likely by preventing premature differentiation. The future use of Ostreococcus tauri and Physcomitrella patens as biological models should allow us to obtain a better insight into the functional importance of WOX13 OG genes.

LFR, which encodes a novel nuclear-localized Armadillo-repeat protein, affects multiple developmental processes in the aerial organs in Arabidopsis

The Armadillo (ARM)-repeat domain is a 42-amino acid protein-protein interaction motif present in many eukaryotic proteins. ARM-repeat proteins function in many cellular processes, including cytoskeletal regulation, nucleo-cytoplasmic trafficking, and transcriptional regulation. More than 100 genes encoding ARM-repeat proteins are predicted to exist in the Arabidopsis thaliana genome; however, most of them have unknown biological functions. Using map-based cloning, we isolated a novel recessive loss-of-function mutant, lfr-1, with developmental and morphological defects at the vegetative stage in the cotyledons and true leaves, and during the reproductive phase in the flowers and siliques. Complementation experiments and an analysis of the T-DNA insertion mutant lfr-2 revealed that LFR was responsible for all of the mutant phenotypes. LFR encodes a protein with three putative ARM-repeat domains that tends to cluster in the nucleus as discrete rounded speckles. LFR was broadly expressed while LFR was largely concentrated in the stem apex and root tip. Our data suggest that LFR is a novel nuclear-localized ARM-repeat protein that functions in leaf and flower development in Arabidopsis.

Global identification of DELLA target genes during Arabidopsis flower development.

Gibberellin (GA) plays important roles in regulating many aspects of plant development. GA derepresses its signaling pathway by promoting the degradation of DELLA proteins, a family of nuclear growth repressors. Although the floral organ identity is established in flowers of the GA-deficient mutant ga1-3, the growth of all floral organs is severely retarded. In particular, abortive anther development in ga1-3 results in male sterility. Genetic analysis has revealed that various combinations of null mutants of DELLA proteins could gradually rescue floral organ defects in ga1-3 and that RGA is the most important DELLA protein involved in floral organ development. To elucidate the early molecular events controlled by RGA during flower development, we performed whole-genome microarray analysis to identify genes in response to the steroid-inducible activation of RGA in ga1-3 rgl2 rga 35S:RGA-GR. Although DELLA proteins were suggested as transcriptional repressors, similar numbers of genes were down-regulated or up-regulated by RGA during floral organ development. More than one-third of RGA down-regulated genes were specifically or predominantly expressed in stamens. A significant number of RGA-regulated genes are involved in phytohormone signaling or stress response. Further expression analysis through activation of RGA by steroid induction combined with cycloheximide identified eight genes as immediate targets of RGA. In situ hybridization and transgenic studies further showed that the expression pattern and function of several selected genes were consistent with the predictions from microarray analysis. These results suggest that DELLA regulation of floral organ development is modulated by multiple phytohormones and stress signaling pathways.

Cloning and characterization of 5 MADS-box cDNAs isolated from citrus fruit tissue

Abstract We isolated and characterized 5 MADS-box cDNA clones of CitMADS1 , CitMADS3 , CitMADS5 , CitMADS6 and CitMADS8 from fruit tissues of Satsuma mandarin ( Citrus unshiu Marc.). Sequence analysis revealed that they have high sequence identities with known MADS-box genes and that their predicted proteins possess the general structural features of the M domain and the K domain. In a phylogenetic tree with 24 known Arabidopsis MADS-box genes, 5 Citrus MADS-box genes were classified into 4 clades of the MADS-box gene with independent gene functions. Their broad expression profiles in the Citrus life cycle suggested that they play roles in flower and fruit development. Most of them, except CitMADS1 and CitMADS3, are expressed in seedlings before the phase transition from vegetative to reproductive growth. CitMADS1 and CitMADS3 are not expressed in vegetative organs and could serve as a molecular marker for reproductive development. These results suggest that the regulatory relationships of genes involved in Arabidopsis flower and fruit development are not directly applicable to the homologous genes in Citrus and that different functions might be considered.

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

Floral patterning and morphogenesis are controlled by many transcription factors including floral homeotic proteins, by which floral organ identity is determined. Recent studies have uncovered widespread regulation of transcription factors by microRNAs (miRNAs), approximately 21-nucleotide non-coding RNAs that regulate protein-coding RNAs through transcript cleavage and/or translational inhibition. The regulation of the floral homeotic gene APETALA2 (AP2) by miR172 is crucial for normal Arabidopsis flower development and is likely to be conserved across plant species. Here we probe the activity of the AP2/miR172 regulatory circuit in a heterologous Solanaceae species, Nicotiana benthamiana. We generated transgenic N. benthamiana lines expressing Arabidopsis wild type AP2 (35S::AP2), miR172-resistant AP2 mutant (35S::AP2m3) and MIR172a-1 (35S::MIR172) under the control of the cauliflower mosaic virus 35S promoter. 35S::AP2m3 plants accumulated high levels of AP2 mRNA and protein and exhibited floral patterning defects that included proliferation of numerous petals, stamens and carpels indicating loss of floral determinacy. On the other hand, nearly all 35S::AP2 plants accumulated barely detectable levels of AP2 mRNA or protein and were essentially non-phenotypic. Overall, the data indicated that expression of the wild type Arabidopsis AP2 transgene was repressed at the mRNA level by an endogenous N. benthamiana miR172 homologue that could be detected using Arabidopsis miR172 probe. Interestingly, 35S::MIR172 plants had sepal-to-petal transformations and/or more sepals and petals, suggesting interference with N. benthamiana normal floral homeotic gene function in perianth organs. Our studies uncover the potential utility of the Arabidopsis AP2/miR172 system as a tool for manipulation of floral architecture and flowering time in non-model 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.

Role of auxin in regulating Arabidopsis flower development

To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip.