My favourite flowering image: an Arabidopsis inflorescence expressing fluorescent reporters for the APETALA3 and SUPERMAN genes

When asked to provide a picture for the cover of the Flowering Newsletter, I picked this image of an Arabidopsis thaliana inflorescence expressing fluorescent reporters for two key regulators of flower development: APETALA3 (AP3), which promotes petal and stamen identity, and SUPERMAN (SUP), which encodes a transcriptional repressor that defines the boundary between stamens and pistil (Fig. 1). The choice was easy: it was an important breakthrough in my research on the role of SUP in the separation of stamens in whorl 3 and carpels in whorl 4; and among the images of flowers I have taken with a confocal microscope, it is also one my favourites aesthetically. The image won awards at the 2015 Nikon Small World and FASEB BioArt competitions and is published in Prunet et al. (2017)

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope

My favourite flowering image: an Arabidopsis inflorescence expressing fluorescent reporters for the APETALA3 and SUPERMAN genes

When asked to provide a picture for the cover of the Flowering Newsletter, I picked this image of an Arabidopsis thaliana inflorescence expressing fluorescent reporters for two key regulators of flower development: APETALA3 (AP3), which promotes petal and stamen identity, and SUPERMAN (SUP), which encodes a transcriptional repressor that defines the boundary between stamens and pistil (Fig. 1). The choice was easy: it was an important breakthrough in my research on the role of SUP in the separation of stamens in whorl 3 and carpels in whorl 4; and among the images of flowers I have taken with a confocal microscope, it is also one my favourites aesthetically. The image won awards at the 2015 Nikon Small World and FASEB BioArt competitions and is published in Prunet et al. (2017)

Live confocal imaging of Arabidopsis flower buds

Recent advances in confocal microscopy, coupled with the development of numerous fluorescent reporters, provide us with a powerful tool to study the development of plants. Live confocal imaging has been used extensively to further our understanding of the mechanisms underlying the formation of roots, shoots and leaves. However, it has not been widely applied to flowers, partly because of specific challenges associated with the imaging of flower buds. Here, we describe how to prepare and grow shoot apices of Arabidopsis in vitro, to perform both single-point and time-lapse imaging of live, developing flower buds with either an upright or an inverted confocal microscope

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

Expression of KNUCKLES in the Stem Cell Domain Is Required for Its Function in the Control of Floral Meristem Activity in Arabidopsis

In the model plant Arabidopsis thaliana, the zinc-finger transcription factor KNUCKLES (KNU) plays an important role in the termination of floral meristem activity, a process that is crucial for preventing the overgrowth of flowers. The KNU gene is activated in floral meristems by the floral organ identity factor AGAMOUS (AG), and it has been shown that both AG and KNU act in floral meristem control by directly repressing the stem cell regulator WUSCHEL (WUS), which leads to a loss of stem cell activity. When we re-examined the expression pattern of KNU in floral meristems, we found that KNU is expressed throughout the center of floral meristems, which includes, but is considerably broader than the WUS expression domain. We therefore hypothesized that KNU may have additional functions in the control of floral meristem activity. To test this, we employed a gene perturbation approach and knocked down KNU activity at different times and in different domains of the floral meristem. In these experiments we found that early expression in the stem cell domain, which is characterized by the expression of the key meristem regulatory gene CLAVATA3 (CLV3), is crucial for the establishment of KNU expression. The results of additional genetic and molecular analyses suggest that KNU represses floral meristem activity to a large extent by acting on CLV3. Thus, KNU might need to suppress the expression of several meristem regulators to terminate floral meristem activity efficiently

Genetics and Plant Development

There are only three grand theories in biology: the theory of the cell, the theory of the gene, and the theory of evolution. Two of these, the cell and gene theories, originated in the study of plants, with the third resulting in part from botanical considerations as well. Mendel's elucidation of the rules of inheritance was a result of his experiments on peas. The rediscovery of Mendel's work in 1900 was by the botanists de Vries, Correns, and Tschermak. It was only in subsequent years that animals were also shown to have segregation of genetic elements in the exact same manner as had been shown in plants. The story of developmental biology is different – while the development of plants has long been studied, the experimental and genetic approaches to developmental mechanism were developed via experiments on animals, and the importance of genes in development (e.g., Waddington, 1940) and their use for understanding developmental mechanisms came to botanical science much later – as late as the 1980s

Redundancy in the temporal control of floral meristem termination in Arabidopsis thaliana (functional analysis of three modifiers of crabs claw)

Aerial growth of Arabidopsis thaliana is realized by the shoot apical meristem (SAM), which contains stem cells whose regular divisions sustain the continuous production of new organs. During reproductive development, the SAM produces flower meristems (FMs) on its flanks. Contrary to the SAM that generated them, FMs do not grow indefinitely and produce flowers, which are determinate structures, with a fixed number of organs. This determinacy is due to the repression of WUSHEL (WUS), which confers their identity to stem cells, by the homeotic gene AGAMOUS (AG). This arrest of stem cell maintenance is linked to the female developmental program of the flower, and requires SUPERMAN (SUP), which establish the boundary between the male an female parts of the flower. During my doctorate, I realized a functional analyses of three genes, REBELOTE (RBL), SQUINT (SQN) and ULTRAPETALA1 (ULT1). Combined mutation of two of these genes, or one of them and CRABS CLAW (CRC) triggers a strong loss of FM termination, with numerous supernumerary organs being produced. The range of phenotypes we got suggests that disruption of stem cell maintenance within the FM is a progressive process, which is redundantly controlled by several genes. Genetic and molecular analyses show that our mutant phenotypes result from a down regulation of AG in a sub-domain of its expression pattern, in the centre of FM. However, this decrease of AG expression is insufficient to explain all the phenotypes we observed, and genetic data suggest that RBL, SQN and ULT1 also influence SUP function. Additionally, preliminary analyses support a role for SQN in the CLAVATA pathway, while RBL may influence microRNAs biosynthesis. Finally RBL, SQN and ULT1 seem to contribute to flower homeostasis.La croissance aérienne d Arabidopsis thaliana est assurée par le méristème apical caulinaire (MAC), qui contient des cellules souche dont les divisions permanentes permettent la mise en place continuelle de nouvelles structures. Au cours du développement reproducteur, le MAC produit des méristèmes floraux (MFs) sur ses flancs. Contrairement au MAC et bien qu ils en soient issus, les MFs ne présentent pas de croissance indéfinie et produisent des fleurs, qui sont des structures déterminées, constituées d un nombre fixe d organes. Cette détermination est liée à la répression du gène WUSHEL (WUS), qui confère leur identité aux cellules souche, par le gène homéotique AGAMOUS (AG). Cet arrêt de l entretien des cellules souche au sein du MF est lié à la mise en place des organes femelles de la fleur, les carpelles, et requiert l action de SUPERMAN (SUP), qui permet l établissement de la frontière entre les parties mâle et femelle de la fleur. Le travail de cette thèse consiste en la caractérisation de trois gènes, REBELOTE (RBL), SQUINT (SQN) et ULTRAPETALA1 (ULT1). La mutation combinée de 2 de ces gènes, ou de l un d entre eux et de CRABS CLAW (CRC), entraîne une perte marquée de l arrêt du MF, qui continue alors indéfiniment à produire de nouveaux organes. La gamme de phénotypes obtenus suggère que l arrêt de l entretien des cellules souche au centre du MF est un phénomène progressif, contrôlé de manière redondante par plusieurs gènes. Une analyse génétique et moléculaire montre que les phénotypes obtenus résultent d une baisse d expression d AG dans une partie interne de son domaine d expression, au centre du MF. Cependant, ce défaut d expression d AG est insuffisant pour expliquer tous les phénotypes observés, et les données génétiques obtenues suggèrent que RBL, SQN et ULT1 influencent aussi l activité de SUP. Enfin, des études préliminaires suggèrent que SQN pourrait influencer AG via la voie CLAVATA (CLV), tandis que RBL semble jouer sur la biosynthèse des microARNs, dont une famille, miR172, affecte l activité d AG. Finalement, RBL, SQN et ULT1 semblent contribuer à l homéostasie du développement floral.LYON-ENS Sciences (693872304) / SudocSudocFranceF