Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation

Abstract

BackgroundThe pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana.MethodsHere, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. ResultsDozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone.ConclusionsThe major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses.Electronic supplementary materialThe online version of this article (10.1186/s12870-017-1136-x) contains supplementary material, which is available to authorized users.

LRX Proteins play a crucial role in pollen grain and pollen tube cell wall development

“…A detailed step-by-step description of the protocol used was described previously (Ndinyanka Fabrice et al, 2017). Briefly, PT specimens were fixed in 1.25% glutaraldehyde in 0.05% cacodylate buffer, post-fixed in 2% OsO4, dehydrated in acetone, and then embedded in Epon.…”

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

“…Nevertheless, there are a number of examples for successful labeling of epoxy resins after conventional processing or cryofixation/freeze‐substitution. These include small neurotransmitter labeling (Ottersen, Zhang, & Walberg, ), immunolabeling, and lectin labeling of carbohydrate epitopes (Moussian et al, ; Ndinyanka Fabrice et al, ; Richter et al, ), immunolabeling of proteins (Groos, Reale, & Luciano, ), biotin of biotinylated proteins (Viens et al, ), glycoinositolphospholipids, and glycosphingolipids (Winter, Fuchs, McConville, Stierhof, & Overath, ). The experiments shown in this study, but also in previous studies from our laboratory demonstrated that Epon sections can be superior for many immunolabeling studies, at least when studying archaeal cells and microalgal cells (Heimerl et al, ; Küper et al, ; Liu et al, ; Mayer et al, ; Meyer, Heimerl, Wirth, Klingl, & Rachel, ; Mix et al, ; Peschke et al, ; Schreiber et al, ).…”

Defects in Cell Wall Differentiation of the Arabidopsis Mutant rol1-2 Is Dependent on Cyclin-Dependent Kinase CDK8

“…Serial dehydration was carried out in increasing concentrations (for 10 min each, 30%, 50%, 70%, 90% and 2× in 100% v/v ) of acetone in water, the roots were infiltrated overnight with 50% v/v Epon/acetone, and then embedded in 100% Epon resin. A very detailed step-by-step description has been previously published [ 25 ]. For immunolabelling, ultrathin sections produced of material embedded as described above were incubated overnight with 1:150 dilution of the anti-(1,3)-β-glucan antibody against callose (Biosupplies, Bundoora, Australia) in 4% nonfatted milk in PBS buffer (pH 7.2).…”