Actin cytoskeleton regulates pollen tube growth and tropism

La fertilisation chez les plantes dépend de la livraison des cellules spermatiques contenues dans le pollen à l’ovule. Au contact du stigmate, le grain de pollen s’hydrate et forme une protubérance, le tube pollinique, chargé de livrer les noyaux spermatiques à l’ovule. Le tube pollinique est une cellule à croissance rapide, anisotrope et non autotrophe; ainsi tout au long de sa croissance à travers l’apoplaste du tissu pistillaire, le tube pollinique puise ses sources de carbohydrates et de minéraux du pistil. Ces éléments servent à la synthèse des constituants de la paroi qui seront acheminés par des vésicules de sécrétion jusqu’à l’apex du tube. Ce dernier doit aussi résister à des pressions mécaniques pour maintenir sa forme cylindrique et doit répondre à différents signaux directionnels pour pouvoir atteindre l’ovule. Mon projet de doctorat était de comprendre le rôle du cytosquelette dans la croissance anisotrope du tube pollinique et d’identifier les éléments responsables de sa croissance et de son guidage. Le cytosquelette du tube pollinique est composé des microfilaments d’actine et des microtubules. Pour assurer une bonne croissance des tubes polliniques in vitro, les carbohydrates et les éléments de croissance doivent être ajoutés au milieu à des concentrations bien spécifiques. J’ai donc optimisé les conditions de croissance du pollen d’Arabidopsis thaliana et de Camellia japonica qui ont été utilisés avec le pollen de Lilium longiflorum comme modèles pour mes expériences. J’ai développé une méthode rapide et efficace de fixation et de marquage du tube pollinique basée sur la technologie des microondes. J’ai aussi utilisé des outils pharmacologiques, mécaniques et moléculaires couplés à différentes techniques de microscopie pour comprendre le rôle du cytosquelette d’actine lors de la croissance et le tropisme du tube pollinique. J’ai trouvé que le cytosquelette d’actine et plus précisément l’anneau d’actine localisé dans la partie sub-apicale du tube est fortement impliqué dans la croissance et le maintien de l’architecture du tube à travers le contrôle de la livraison des vésicules de sécrétion. J’ai construit une chambre galvanotropique qui peut être montée sur un microscope inversé et qui sert à envoyer des signaux tropistiques bien précis à des tubes polliniques en croissance. J’ai trouvé que les filaments d’actine sont impliqués dans la capacité du tube pollinique à changer de direction. Ce comportement tropistique dépend de la concentration du calcium dans le milieu de croissance et du flux de calcium à travers des canaux calciques. Le gradient de calcium établi dans le tube pollinique affecte l’activité de certaines protéines qui se lient à l’actine et dont le rôle est la réorganisation des filaments d’actine. Parmi ces protéines, il y a celles de dépolymérisation de l’actine (ADF) dont deux spécifiquement exprimées dans le gamétophyte mâle d’Arabidopsis (ADF7 et ADF10). Par marquage avec des proteins fluorescents, j’ai trouvé que l’ADF7 et l’ADF10 ont des expressions différentielles pendant la microsporogenèse et la germination et croissance du tube pollinique et qu’elles partagent entre elles des rôles importants durant ces différents stades.Fertilization in plants depends on the delivery of the sperm cells in the pollen grain through the pollen tube to the ovule. The pollen tube is a highly anisotropic, fast growing cellular protuberance. Because the pollen tube is non autotrophic, it requires a steady supply of carbohydrates and minerals supplied by the pistil to sustain its growth. These elements serve for the synthesis of cell wall material, delivered to the site of cell wall assembly in secretory vesicles that are transported along the actin cytoskeleton and deposited at the growing apex of the tube. The tube has to resist external deformation forces in order to maintain its cylindrical shape and to respond to various directional signals in order to reach its target. My objectives were to identify the role of the cytoskeleton in the anisotropic growth of the pollen tube and to determine how the tube responds to directional cues. The cytoskeleton in the pollen tube consists of microfilaments and microtubules, both forming long filamentous elements. For in vitro growing pollen tubes, carbohydrates and growth minerals have to be added to the growth medium in specific amounts order to sustain pollen tube growth. I optimized the growth conditions of Arabidopsis thaliana and Camellia japonica pollen tubes which, in addition to pollen from Lilium longiflorum, were used as model species for my experiments. I developed a microwave based, fast and efficient fixation and labelling protocol for pollen tubes. I used pharmacological, mechanical, molecular and microscopical tools to study the role of the cytoskeleton in pollen tube growth and tropism. I found that the actin cytoskeleton, and more specifically the subapical actin fringe, plays an important role in the regulation of pollen tube growth and architecture through the controlled delivery of secretory vesicles to the growing apex. I constructed a galvanotropic chamber that can be mounted on an inverted microscope to induce controlled tropic triggers. I found that the actin cytoskeleton is also involved in the ability of the pollen tube to change its direction. This tropic behaviour was shown to be dependent on the concentration of calcium ions in the growth medium and calcium influx through calcium channels. The cytosolic calcium gradient in the pollen tube regulates the activity of various actin binding proteins that are responsible for remodelling the actin cytoskeleton. Among these proteins are two Arabidopsis gametophyte-specific actin depolymerizing factors (ADFs) that I tagged with two intrinsically fluorescent proteins. I found that ADF7 and ADF10 are differentially expressed during microsporogenesis and pollen tube germination and growth and that they likely divide important functions between them

How to let go: pectin and plant cell adhesion.

This is the final version of the article. It first appeared from Frontiers via http://dx.doi.org/10.3389/fpls.2015.00523Plant cells do not, in general, migrate. They maintain a fixed position relative to their neighbors, intimately linked through growth and differentiation. The mediator of this connection, the pectin-rich middle lamella, is deposited during cell division and maintained throughout the cell's life to protect tissue integrity. The maintenance of adhesion requires cell wall modification and is dependent on the actin cytoskeleton. There are developmental processes that require cell separation, such as organ abscission, dehiscence, and ripening. In these instances, the pectin-rich middle lamella must be actively altered to allow cell separation, a process which also requires cell wall modification. In this review, we will focus on the role of pectin and its modification in cell adhesion and separation. Recent insights gained in pectin gel mechanics will be discussed in relation to existing knowledge of pectin chemistry as it relates to cell adhesion. As a whole, we hope to begin defining the physical mechanisms behind a cells' ability to hang on, and how it lets go.The writing of this review was carried out with the help of grant BB-L002884-1 (BBSRC, UK)

Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry.

Fast directional growth is a necessity for the young seedling; after germination, it needs to quickly penetrate the soil to begin its autotrophic life. In most dicot plants, this rapid escape is due to the anisotropic elongation of the hypocotyl, the columnar organ between the root and the shoot meristems. Anisotropic growth is common in plant organs and is canonically attributed to cell wall anisotropy produced by oriented cellulose fibers. Recently, a mechanism based on asymmetric pectin-based cell wall elasticity has been proposed. Here we present a harmonizing model for anisotropic growth control in the dark-grown Arabidopsis thaliana hypocotyl: basic anisotropic information is provided by cellulose orientation) and additive anisotropic information is provided by pectin-based elastic asymmetry in the epidermis. We quantitatively show that hypocotyl elongation is anisotropic starting at germination. We present experimental evidence for pectin biochemical differences and wall mechanics providing important growth regulation in the hypocotyl. Lastly, our in silico modelling experiments indicate an additive collaboration between pectin biochemistry and cellulose orientation in promoting anisotropic growth

Data for Bou Daher at al. 2018

This .zip file contains all raw experimental data associated with the publication of record (produced by Bou Daher, Chen and Braybrook). Data organization is by Figure, then by data type. Details in the README file

High Light Intensity Leads to Increased Peroxule-Mitochondria Interactions in Plants

Peroxules are thin protrusions from spherical peroxisomes produced under low levels of reactive oxygen species (ROS) stress. Whereas stress mitigation favours peroxule retraction, prolongation of the ROS stress leads to the elongation of the peroxisome into a tubular form. Subsequently, the elongated form becomes constricted through the binding of proteins such as dynamin related proteins 3A and 3B and eventually undergoes fission to increase the peroxisomal population within a cell. The events that occur in the short time window between peroxule initiation and the tubulation of the entire peroxisome have not been observed in living plant cells. Here, using fluorescent protein aided live-imaging, we show that peroxules are formed after only 4 minutes of high light (HL) irradiation during which there is a perceptible increase in the cytosolic levels of hydrogen peroxide. Using a stable, double transgenic line of Arabidopsis thaliana expressing a peroxisome targeted YFP and a mitochondrial targeted GFP probe, we observed sustained interactions between peroxules and small, spherical mitochondria. Further it was observed that the frequency of HL-induced interactions between peroxules and mitochondria increased in the Arabidopsis anisotropy1 mutant that has reduced cell wall crystallinity and where we show accumulation of higher H2O2 levels than wild type plants. Our observations suggest a testable model whereby peroxules act as interaction platforms for ROS-distressed mitochondria that may release membrane proteins and fission factors. These proteins might thus become easily available to peroxisomes and facilitate their proliferation for enhancing the ROS-combating capability of a plant cell