Arabidopsis Flower Development Research Articles

The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

A global view of transcriptome dynamics during flower development in Capsicum annuum L.

An analysis of genome-wide gene expression profiles during floral organ development can provide important clues about the molecular basis of gene functions and developmental processes. In this study, we analyzed the transcriptome data of 36 samples obtained during floral organ development from pepper ‘6421’ and detected 30 016 genes that were expressed in at least one sample. K-means clustering analysis was used to classify the data into 16 clusters based on the similarities between the dynamic expression profiles of genes. Of these, 15 clusters exhibited notable up-regulation or down-regulation trends in different developmental stages or tissues of floral organs. We identified transcription factors expressed at the early, medium, and late stages of bud development (F1, F5, F9). Transcription factor families such as AP2-ERF, MADS-box, MYB, bHLH, and NAC showed significant levels of enrichment. In comparison with genes expressed in vegetative tissues at different stages, certain genes were specifically up-regulated during flower development; among these, the number of genes specifically up-regulated during the stamen (Sta10) and bud tetrad development (F4) stages was the highest. Through extensive studies of the ABCDE model of flower development in Arabidopsis, we identified 17 ABCDE model candidate genes in pepper, most of which were up-regulated at specific stages of flower bud development. The expression data provided in this study is the most comprehensive dataset available for pepper to date and will serve as a resource for identifying the functions of many specific genes involved in flower development in pepper and other Solanaceae plants.

Hormones and Flower Development in Arabidopsis.

Sexual reproduction requires the participation of two gametes, female and male. In angiosperms, gametes develop in specialized organs, pollen (containing the male gametes) develops in the stamens, and the ovule (containing the female gamete) develops in the gynoecium. In Arabidopsis thaliana, the female and male sexual organs are found within the same structure called flower, surrounded by the perianth, which is composed of petals and sepals. During flower development, different organs emerge in an established order and throughout their development distinct tissues within each organ are differentiated. All this requires the coordination and synchronization of several biological processes. To achieve this, hormones and genes work together. These components can interact at different levels generating hormonal interplay and both positive and negative feedback loops, which in turn, gives robustness, stability, and flexibility to flower development. Here, we summarize the progress made on elucidating the role of different hormonal pathways during flower development in Arabidopsis thaliana.

Flower Development in Arabidopsis.

Like in other angiosperms, the development of flowers in Arabidopsis starts right after the floral transition, when the shoot apical meristem (SAM) stops producing leaves and makes flowers instead. On the flanks of the SAM emerge the flower meristems (FM) that will soon differentiate into the four main floral organs, sepals, petals, stamens, and pistil, stereotypically arranged in concentric whorls. Each phase of flower development-floral transition, floral bud initiation, and floral organ development-is under the control of specific gene networks. In this chapter, we describe these different phases and the gene regulatory networks involved, from the floral transition to the floral termination.

Floral Induction Systems for the Study of Arabidopsis Flower Development.

Assessing the molecular changes that occur over the course of flower development is hampered by difficulties in isolating sufficient amounts of floral tissue at specific developmental stages. This is especially problematic when investigating molecular events at early stages of Arabidopsis flower development, as floral buds are minute and are initiated sequentially so that a single flower on an inflorescence is at a given developmental stage. Moreover, young floral buds are hidden by older flowers, which presents an additional challenge for dissection. To circumvent these issues, floral induction systems that allow the simultaneous induction of a large number of flowers on the inflorescence of a single plant were developed. To allow the plant community to avail of the full benefits of these systems, we address some common problems that can be encountered when growing these plants and collecting floral buds for analysis.

Genome-Wide Analysis of Snf2 Gene Family Reveals Potential Role in Regulation of Spike Development in Barley

Sucrose nonfermenting 2 (Snf2) family proteins, as the catalytic core of ATP-dependent chromatin remodeling complexes, play important roles in nuclear processes as diverse as DNA replication, transcriptional regulation, and DNA repair and recombination. The Snf2 gene family has been characterized in several plant species; some of its members regulate flower development in Arabidopsis. However, little is known about the members of the family in barley (Hordeum vulgare). Here, 38 Snf2 genes unevenly distributed among seven chromosomes were identified from the barley (cv. Morex) genome. Phylogenetic analysis categorized them into 18 subfamilies. They contained combinations of 21 domains and consisted of 3 to 34 exons. Evolution analysis revealed that segmental duplication contributed predominantly to the expansion of the family in barley, and the duplicated gene pairs have undergone purifying selection. About eight hundred Snf2 family genes were identified from 20 barley accessions, ranging from 38 to 41 genes in each. Most of these genes were subjected to purification selection during barley domestication. Most were expressed abundantly during spike development. This study provides a comprehensive characterization of barley Snf2 family members, which should help to improve our understanding of their potential regulatory roles in barley spike development.

MicroRNA172 controls inflorescence meristem size through regulation of APETALA2 in Arabidopsis.

The APETALA2 (AP2) transcription factor regulates flower development, floral transition and shoot apical meristem (SAM) maintenance in Arabidopsis. AP2 is also regulated at the post-transcriptional level by microRNA172 (miR172), but the contribution of this to SAM maintenance is poorly understood. We generated transgenic plants carrying a form of AP2 that is resistant to miR172 (rAP2) or carrying a wild-type AP2 susceptible to miR172. Phenotypic and genetic analyses were performed on these lines and mir172 mutants to study the role of AP2 regulation by miR172 on meristem size and the rate of flower production. We found that rAP2 enlarges the inflorescence meristem by increasing cell size and cell number. Misexpression of rAP2 from heterologous promoters showed that AP2 acts in the central zone (CZ) and organizing center (OC) to increase SAM size. Furthermore, we found that AP2 is negatively regulated by AUXIN RESPONSE FACTOR 3 (ARF3). However, genetic analyses indicated that ARF3 also influences SAM size and flower production rate independently of AP2. The study identifies miR172/AP2 as a regulatory module controlling inflorescence meristem size and suggests that transcriptional regulation of AP2 by ARF3 fine-tunes SAM size determination.

High spatial resolution imaging of the dynamics of cuticular lipid deposition during Arabidopsis flower development.

The extensive collection of glossy (gl) and eceriferum (cer) mutants of maize and Arabidopsis have proven invaluable in dissecting the branched metabolic pathways that support cuticular lipid deposition. This bifurcated pathway integrates a fatty acid elongation‐decarbonylative branch and a fatty acid elongation‐reductive branch, which collectively has the capacity to generate hundreds of cuticular lipid metabolites. In this study, a combined transgenic and biochemical strategy was implemented to explore and compare the physiological function of three homologous genes, Gl2, Gl2‐like, and CER2, in the context of this branched pathway. These biochemical characterizations integrated new extraction chromatographic procedures with high spatial resolution mass spectrometric imaging methods to profile the cuticular lipids on developing floral tissues transgenically expressing these transgenes in wild‐type or cer2 mutant lines of Arabidopsis. Collectively, these datasets establish that both the maize Gl2 and Gl2‐like genes are functional homologs of the Arabidopsis CER2 gene. In addition, the dynamic distribution of cuticular lipid deposition follows distinct floral organ localization patterns indicating that the fatty acid elongation‐decarbonylative branch of the pathway is differentially localized from the fatty acid elongation‐reductive branch of the pathway.

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development.

Dual-color 3D-dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei.

To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant-specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novel ROXY1 function in the root meristem. Root cells that lack chlorophyll reducing plant-specific background problems that can hamper colocalization 3D microscopy. Thus far, a super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-dSTORM) approach has mainly been applied in animal studies. We established 3D-dSTORM using the roxy1 mutant complemented with green fluorescence protein-ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D-dSTORM was coupled with the coordinate-based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H2 O2 treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual-color 3D-dSTORM/coordinate-based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50nm, which will enable the detection of novel subcellular protein associations and functions.

Ectopic Expression of a Pak-choi YABBY Gene, BcYAB3, Causes Leaf Curvature and Flowering Stage Delay in Arabidopsis thaliana.

The YABBY family are a group of seed plant-specific transcription factors, which are involved in the specification of abaxial polarity in lateral organs. In Arabidopsis thaliana, YABBY3 (YAB3) plays a critical role in regulating abaxial patterning, growth of lateral organs, and inflorescence phyllotaxy. In this study, the BcYAB3 gene was isolated from Pak-choi (Brassica rapa subsp. chinensis). The tissue-specific expression analysis indicated that the BcYAB3 gene has significantly high transcript levels in stem, leaf, and flower. We investigated the subcellular localization of BcYAB3 and found the protein to be expressed in the nucleus. In the transgenic Arabidopsis thaliana plants expressing the BcYAB3 gene, the leaves were curling downward with the plant growth, and the bolting and flowering stages were delayed. These results not only validate the function of BcYAB3 in the leaf and flower development in Arabidopsis, but also contribute to unravel the molecular regulatory mechanism of YAB3 gene in the establishment of adaxial–abaxial polarity of the lateral organs in Pak-choi.

Dynamic control of enhancer activity drives stage-specific gene expression during flower morphogenesis

Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis. H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis. We provide computational and experimental evidence to confirm that distal DNase І hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers.

RGL2 controls flower development, ovule number and fertility in Arabidopsis

DELLA proteins are a group of plant specific GRAS proteins of transcriptional regulators that have a key role in gibberellin (GA) signaling. In Arabidopsis, the DELLA family is formed by five members. The complexity of this gene family raises the question on whether single DELLA proteins have specific or overlapping functions in the control of several GA-dependent developmental processes. To better understand the roles played by RGL2, one of the DELLA proteins in Arabidopsis, two transgenic lines that express fusion proteins of Venus-RGL2 and a dominant version of RGL2, YPet-rgl2Δ17, were generated by recombineering strategy using a genomic clone that contained the RGL2 gene. The dominant YPet-rgl2Δ17 protein is not degraded by GAs, and therefore it blocks the RGL2-dependent GA signaling and hence RGL2-dependent development. The RGL2 role in seed germination was further confirmed using these genetic tools, while new functions of RGL2 in plant development were uncovered. RGL2 has a clear function in the regulation of flower development, particularly stamen growth and anther dehiscence, which has a great impact in fertility. Moreover, the increased ovule number in the YPet-rgl2Δ17 line points out the role of RGL2 in the determination of ovule number.

Genome-wide dynamic network analysis reveals a critical transition state of flower development in Arabidopsis

BackgroundThe flowering transition which is controlled by a complex and intricate gene regulatory network plays an important role in the reproduction for offspring of plants. It is a challenge to identify the critical transition state as well as the genes that control the transition of flower development. With the emergence of massively parallel sequencing, a great number of time-course transcriptome data greatly facilitate the exploration of the developmental phase transition in plants. Although some network-based bioinformatics analyses attempted to identify the genes that control the phase transition, they generally overlooked the dynamics of regulation and resulted in unreliable results. In addition, the results of these methods cannot be self-explained.ResultsIn this work, to reveal a critical transition state and identify the transition-specific genes of flower development, we implemented a genome-wide dynamic network analysis on temporal gene expression data in Arabidopsis by dynamic network biomarker (DNB) method. In the analysis, DNB model which can exploit collective fluctuations and correlations of different metabolites at a network level was used to detect the imminent critical transition state or the tipping point. The genes that control the phase transition can be identified by the difference of weighted correlations between the genes interested and the other genes in the global network. To construct the gene regulatory network controlling the flowering transition, we applied NARROMI algorithm which can reduce the noisy, redundant and indirect regulations on the expression data of the transition-specific genes. In the results, the critical transition state detected during the formation of flowers corresponded to the development of flowering on the 7th to 9th day in Arabidopsis. Among of 233 genes identified to be highly fluctuated at the transition state, a high percentage of genes with maximum expression in pollen was detected, and 24 genes were validated to participate in stress reaction process, as well as other floral-related pathways. Composed of three major subnetworks, a gene regulatory network with 150 nodes and 225 edges was found to be highly correlated with flowering transition. The gene ontology (GO) annotation of pathway enrichment analysis revealed that the identified genes are enriched in the catalytic activity, metabolic process and cellular process.ConclusionsThis study provides a novel insight to identify the real causality of the phase transition with genome-wide dynamic network analysis.

Meristem Termination and Organ Number Control in Early Stage of Arabidopsis Flower Development

Meristem Termination and Organ Number Control in Early Stage of Arabidopsis Flower Development

Investigation of the Effect of AtWIN1/SHN1 Overexpression on Poplar Trees

Background: Interactions between plants and the environment occur primarily at the leaf level. The plant cuticle consists of a menagerie of lipids, waxes and polymers merging to form an insoluble membrane to protect plant leaves from contamination. In Arabidopsis, wax Inducer1/shine1 (WIN1/SHN1) and its family members have demonstrated roles in wax biosynthesis and cutin formation, the primary component of the cuticle layer composition. Constitutive overexpression of the Arabidopsis WIN1/SHN1 (wax inducer1, shine1, AtWIN1/SHN1) gene has led to improved water stress tolerance, altered stomatal densities and morphological changes in leaf and flower development in Arabidopsis. Other expression studies using the WIN1/SHN1 gene have shown heightened defense responses and malformations of the cuticle. Materials and Methods: Constructs of AtWIN1/SHN1 were developed and used to genetically transform poplar trees that were later analyzed to verify presence of the construct. Results: A total of five transgenic lines with 100 ramets each were generated for water-use efficiency testing. All lines displayed glossy leaves, decreased stomatal densities and improved water-use efficiencies. Several lines presented similar phenotypes and water-use efficiencies but others were unique. Conclusion: This study used an inventive method to generate ornamental trees with improved water-use efficiencies as a proactive method for protecting water resources and resisting drought.

BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance.

Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors regulate plant responses to the environment in large part by controlling the abundance of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. However, the molecular determinants of this essential signaling mechanism still remain largely unknown. Here, we present evidence that the BLADE-ON-PETIOLE (BOP) genes, which have previously been shown to control leaf and flower development in Arabidopsis, are involved in controlling the abundance of PIF4. Genetic analysis shows that BOP2 promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing PIF4 activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the bop2 mutant. Moreover, we found that BOP proteins physically interact with both PIF4 and CULLIN3A and that a CULLIN3-BOP2 complex ubiquitinates PIF4 in vitro. This shows that BOP proteins act as substrate adaptors in a CUL3BOP1/BOP2 E3 ubiquitin ligase complex, targeting PIF4 proteins for ubiquitination and subsequent degradation.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1.

BackgroundThe ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown.ResultsHere we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type, but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development.ConclusionsOur results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0571-9) contains supplementary material, which is available to authorized users.

Isolation and expression analysis of three different flowering genes(TtLFY, TtAP1, and TtAP2) from an unusual legume species, Thermopsis turcica

LEAFY (LFY), APETALA1 (AP1), and APETALA2 (AP2) genes encode three different transcription factors that control and regulate flower initiation and development in Arabidopsis. By using 3'- and 5'-RACE analysis, we isolated and sequentially characterized TtLFY (a LEAFY-like gene), TtAP1 (a MADS-box-like gene), and TtAP2 (an AP2/ERBF-like gene) in Thermopsis turcica, an unusual endemic legume species with three free carpellated flower structure. Semiquantitative RT-PCR analysis for 18 different vegetative and reproductive tissues of T. turcica indicated TtLFY transcripts mainly in the shoot tips and young floral buds and TtAP1 transcripts in the sepals and petals; however, TtAP2 transcripts were detected in all tissues. This is the first record for a LFY-like gene, TtLFY, expressed in the shoot tips of an underground plant section and for an AP2-like gene transcript found in all tissues, similar to a housekeeping gene.