Cytoskeleton Remodeling in Arabidopsis Stigmatic Cells Following Pollination.

In plants, the first interaction that occurs between the male gametophytes (pollen grains) and the stigmatic epidermis of the female organ is crucial for successful reproduction. The stigma consists of a dome of flask-shaped cells specialized in pollen capture. In these stigmatic cells, the cytoskeleton network (cortical microtubules and actin microfilaments) actively responds to pollen contact and undergoes dynamic remodeling required for successful pollen acceptance to occur. Here, we have designed several microscopy mountings to monitor stigmatic cytoskeleton dynamics. These designs are based on the constraints linked to the tightly regulated pollen-stigma interaction and depend upon the experimental goal, either a static view or live-cell imaging.

Sculpting the surface: Structural patterning of plant epidermis.

Plant epidermis are multifunctional surfaces that directly affect how plants interact with animals or microorganisms and influence their ability to harvest or protect from abiotic factors. To do this, plants rely on minuscule structures that confer remarkable properties to their outer layer. These microscopic features emerge from the hierarchical organization of epidermal cells with various shapes and dimensions combined with different elaborations of the cuticle, a protective film that covers plant surfaces. Understanding the properties and functions of those tridimensional elements as well as disentangling the mechanisms that control their formation and spatial distribution warrant a multidisciplinary approach. Here we show how interdisciplinary efforts of coupling modern tools of experimental biology, physics, and chemistry with advanced computational modeling and state-of-the art microscopy are yielding broad new insights into the seemingly arcane patterning processes that sculpt the outer layer of plants.

KATANIN and cortical microtubule organization have a pivotal role in early pollen tube guidance.

Following pollen deposition on the receptive surface of the stigma, pollen germinates a tube that carries male gametes toward the ovule where fertilization occurs. As soon as it emerges from the pollen grain, the pollen tube has to be properly guided through the pistil tissues so as to reach the ovule and ensure double fertilization. Chemical attractants, nutrients as well as receptor kinase-dependent signaling pathways have been implicated in this guidance. Recently, we showed in Arabidopsis that the microtubule severing enzyme KATANIN, by acting both on cortical microtubule (CMT) dynamics and cellulose microfibril (CMF) deposition, conferred particular mechanical properties to the papilla cell wall that act as active guidance factors. Here we confirm the importance of KATANIN and CMT orientation in pollen tube directionality by examining another katanin mutant.

The molecular signatures of compatible and incompatible pollination in Arabidopsis.

BACKGROUND: Fertilization in flowering plants depends on the early contact and acceptance of pollen grains by the receptive papilla cells of the stigma. Deciphering the specific transcriptomic response of both pollen and stigmatic cells during their interaction constitutes an important challenge to better our understanding of this cell recognition event. RESULTS: Here we describe a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. This strategy allowed us to distinguish 80% of transcripts according to their parental origins. We also developed a tool which predicts male/female specific expression for genes without SNP. We report an unanticipated transcriptional activity triggered in stigma upon incompatible pollination and show that following compatible interaction, components of the pattern-triggered immunity (PTI) pathway are induced on the female side. CONCLUSIONS: Our work unveils the molecular signatures of compatible and incompatible pollinations both at the male and female side. We provide invaluable resource and tools to identify potential new molecular players involved in pollen-stigma interaction.

KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis.

Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes first grow within the cell wall of the papilla cells, applying pressure to the cell. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), which in turn mediate deposition of cellulose microfibrils (CMFs). Here, by combining imaging, genetic and chemical approaches, we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 (ktn1-5) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth. Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall.

Flowering plants produce small particles known as pollen that ­ with the help of the wind, bees and other animals ­ carry male sex cells (sperm) to female sex cells (eggs) contained within flowers. When a grain of pollen lands on the female organ of a flower, called the pistil, it gives rise to a tube that grows through the pistil towards the egg cells at the base. The surface of the pistil is covered in a layer of long cells named papillae. Like most plant cells, the papillae are surrounded by a rigid structure known as the cell wall, which is mainly composed of strands known as microfibrils. The pollen tube exerts pressure on a papilla to allow it to grow through the cell wall towards the base of the pistil. Previous studies have shown that the pistil produces signals that guide pollen tubes to the eggs. However, it remains unclear how pollen tubes orient themselves on the surface of papillae to grow in the right direction through the pistil. Riglet et al. combined microscopy, genetic and chemical approaches to study how pollen tubes grow through the surface of the pistils of a small weed known as Arabidopsis thaliana. The experiments showed that an enzyme called KATANIN conferred mechanical properties to the cell walls of papillae that allowed pollen tubes to grow towards the egg cells, and also altered the orientation of the microfibrils in these cell walls. In A. thaliana plants that were genetically modified to lack KATANIN the pollen tubes coiled around the papillae and sometimes grew in the opposite direction to where the eggs were. KATANIN is known to cut structural filaments inside the cells of plants, animals and most other living things. By revealing an additional role for KATANIN in regulating the mechanical properties of the papilla cell wall, these findings indicate this enzyme may also regulate the mechanical properties of cells involved in other biological processes.

Live-cell imaging of early events following pollen perception in self-incompatible Arabidopsis thaliana.

Early events occurring at the surface of the female organ are critical for plant reproduction, especially in species with a dry stigma. After landing on the stigmatic papilla cells, the pollen hydrates and germinates a tube, which penetrates the cell wall and grows towards the ovules to convey the male gametes to the embryo sac. In self-incompatible species within the Brassicaceae, these processes are blocked when the stigma encounters an incompatible pollen. Based on the generation of self-incompatible Arabidopsis lines and by setting up a live imaging system, we showed that control of pollen hydration has a central role in pollen selectivity. The faster the pollen pumps water from the papilla during an initial period of 10 min, the faster it germinates. Furthermore, we found that the self-incompatibility barriers act to block the proper hydration of incompatible pollen and, when hydration is promoted by high humidity, an additional control prevents pollen tube penetration into the stigmatic wall. In papilla cells, actin bundles focalize at the contact site with the compatible pollen but not with the incompatible pollen, raising the possibility that stigmatic cells react to the mechanical pressure applied by the invading growing tube.