growth of arabidopsis thaliana plants and preparation of flowers for pollen hydration assay

Une méthode améliorée pour mesurer les profils d’hydratation du pollen chez Arabidopsis thaliana

Successful sexual reproduction in angiosperms typically relies on the transfer of intraspecific pollen grains from the anther to the stigma, either within or between individuals (i.e. pollination). This transfer of pollen grains to a receptive flower is usually mediated by pollinators or abiotic factors; as such, this also frequently results in the deposition of heterospecific pollen under natural conditions. With a few exceptions, the progression of pollination by heterospecific pollen is evolutionarily disadvantageous, reducing reproductive fitness through lost mating opportunities, with most resulting hybrid progeny either failing to develop appropriately or being sterile1. Thus, mechanisms have evolved to block pollination by &#39;incompatible&#39; heterospecific pollen2. Rapid recognition of compatible pollen is therefore arguably the most important process in the early stages of sexual reproduction in many flowering plants.
In the Brassicaceae family, where stigmas are of the &#39;dry&#39; type, a series of molecular checkpoints act at multiple stages in the reproductive process regulating pollination, such that only compatible pollen is successful. Pollen hydration is one of the most important checkpoints (Figure 1), as pollen that fails to hydrate cannot progress to produce a pollen tube and subsequently deliver sperm to the female gametophyte. Frequently, incompatible grains fail to pass this first pollination checkpoint, and thus do not gain access to stigmatic water3. Amongst members of the Brassicaceae family, the recognition of pollen occurs rapidly, with compatibility being established within minutes of pollen grain attachment to the pistil4,5. In recent years, much progress has been made, and we are now beginning to understand the molecular mechanisms that regulate key pollination checkpoints.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65280/65280fig01.jpg" /> 
Figure 1: Overview of key events during compatible pollination. These stages, such as pollen hydration and pollen tube germination, are also pollination &#39;checkpoints&#39; that must be successfully navigated to effect compatible pollination. The diagram represents a &#39;dry&#39; type stigma, which is typical to species from the Brassicaceae family2,20. <a href="https://www.jove.com/files/ftp_upload/65280/65280fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Pioneering research on the Brassica self-incompatibility (SI) system, where &#39;self&#39; pollen is recognized and rejected, established the paradigm for pollen-stigma recognition in Brassicaceae6,7,8,9,10. SI in Brassica and its relatives is mediated by &#39;recognition&#39; proteins that reside on the surface of pollen and at the stigmatic plasma membrane that, upon interaction, lead to pollen rejection. SI pollen rejection operates by disruption of the basal pollen-stigma compatibility system which, when fully activated by the perception of compatible pollen, leads to targeted secretion by the stigma, thus driving pollen hydration (for reviews of the pollen compatibility mechanism, see11,12). In the example of SI, the pollen-borne ligand is a small cysteine-rich protein, S-locus cysteine rich (SCR/SP11), and the stigmatic receptor is the S-locus receptor kinase (SRK).
Recently, in Arabidopsis thaliana, another group of small cysteine-rich pollen-borne proteins, pollen coat protein class Bs (AtPCP-Bs), have been found to be important regulators of pollen acceptance through the activation of pollen hydration13. Stigmatic receptors of the AtPCP-Bs and aspects of the downstream regulatory pathway have also recently been described14,15. Interestingly, mutational studies of genes encoding potential pollen-borne and stigmatic signaling mediators of pollen hydration (including AtPCP-Bs) have failed to generate plants that have a complete block to the pollen hydration checkpoint. This strongly suggests that multiple other, yet undiscovered, factors play a role in the regulation of pollen hydration. Building upon the method first described by Wang et al.13, here we describe an improved high-resolution in vivo bioassay suitable for the identification of subtle pollen hydration defects in candidate mutant A. thaliana lines.

Pleins feux sur l’auteur : Un essai biologique d’hydratation du pollen in vivo à grain unique à haute résolution pour Arabidopsis thaliana  

Un essai biologique in vivo à haute résolution sur l’hydratation du pollen à grain unique pour Arabidopsis thaliana

Watch this Scientific Journal Video about Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay at JoVE.com

Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay   

Croissance des plantes d’Arabidopsis thaliana et préparation des fleurs pour le dosage de l’hydratation du pollen

Commencez par stratifier les graines d’A.thaliana dans de l’eau stérile Milli-Q pendant trois jours à quatre degrés Celsius. Transférer les graines stratifiées dans les pots de compost en pipetant les graines stratifiées. Placez les pots dans une chambre de croissance à environnement contrôlé pendant environ six semaines jusqu’à ce que les inflorescences soient bien établies.Pour assurer une floraison synchrone, semer ensemble les plantes donneuses et réceptrices de pollen et toute autre lignée de plantes témoins appropriées. Un jour avant le bioessai, sélectionnez ces boutons floraux de stade 12 de la plante receveuse, ayant des boutons floraux non ouverts qui montreront l’ouverture de la fleur et la déhiscence de l’anthère le lendemain. Ensuite, placez une lame de verre sous un microscope à dissection stéréo.Collez la région de la tige près de la fleur réceptrice sélectionnée sur cette lame de verre. À l’aide d’une paire de pinces à pointe fine, ouvrez soigneusement le bouton floral et retirez tous les pétales et anthères des fleurs. Une fois cela fait, assurez-vous que le pistil n’est pas endommagé et que le stigmate est exempt de pollen contaminant.

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Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay

619 vues. Commencez par stratifier les graines d’A.thaliana dans de l’eau stérile Milli-Q pendant trois jours à quatre degrés Celsius. Transférer les graines stratifiées dans les pots de compost en pipetant les graines stratifiées. Placez les pots dans une chambre de croissance à environnement contrôlé pendant environ six semaines jusqu’à ce que les inflorescences soient bien établies.Pour assurer une floraison synchrone, semer ensemble les plantes donneuses et réceptrices de p...

Vidéo: Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay   

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...