Live Cell and Immuno-Labeling Techniques to Study Gravitational Effects on Single Plant Cells

Abstract

The constant force of gravity plays a primordial role in the ontogeny of all living organisms. Plants, for example, develop their roots and shoots in accordance with the direction of the gravitational vector. Any change in the magnitude and/or the direction of gravity has an important impact on the development of tissues and cells. In order to understand how the gravitational force affects plant cell growth and differentiation, we established two complementary experimental procedures with which the effect of hyper-gravity on single plant cell development can be assessed. The single model cell system we used is the pollen tube or male gametophyte which, because of its rapid growth behavior, is known for its instant response to external stresses. The physiological response of the pollen tube can be assessed in a quantitative manner based on changes in the composition and spatial distribution of its cell wall components and in the precisely defined pattern of its very dynamic cytoplasmic streaming. Here, we provide a detailed description of the steps required for the immuno-localization of various cell wall components using microwave-assisted techniques and we explain how live imaging of the intracellular traffic can be achieved under hyper-gravity conditions.

Fluorescence visualization of cellulose and pectin in the primary plant cell wall

“…Approximately 5 mg pollen was added to 4 mL of growth medium in Erlenmeyer flasks and incubated at 25 °C under continuous slow shaking. For an illustrated guide on steps for pollen germination, refer to Chebli and Geitmann (2015).…”

“…The fixation was performed for 40 s, followed by three washes with Pipes buffer. For an illustrated guide to the fixation steps performed here refer to Chebli and Geitmann (2015).…”

“…Following the labelling procedure, the samples were mounted on glass slides in a drop of Citifluor antifade AF1 (Electron Microscopy Sciences) for microscopic observation. For an illustrated guide to these steps refer to Chebli and Geitmann (2015). Tables 2 and 3 summarize the probes used here, their details and their targets.…”