Protocol

Four-Dimensional Imaging of Plant Cells During the Cell Cycle

Adapted from Live Cell Imaging, 2nd edition (ed. Goldman et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2010.

INTRODUCTION

Live cell imaging is an essential approach for studying the structure, dynamics, and functions of cells in a living plant under normal or stressed growth conditions. The tiny flowering plant, Arabidopsis thaliana, provides an ideal system to apply various live microscopy techniques. Its small size allows fluorescent light to penetrate the tissues, and its plantlets contain different cell types with different ploidy levels and differentiation stages. Its 2C nucleus contains only five pairs of chromosomes in which heterochromatin domains are organized as chromocenters, and these domains are easily resolved under the microscope. In addition, the availability of powerful genetic tools facilitates the investigation of the molecular mechanisms underlying various cellular phenomena. In designing live imaging experiments, one must keep in mind that plants sense light, temperature, osmolarity, humidity, gravity, and nutrition. In addition, plants also have strong circadian rhythms of physiological behavior and gene expression. Moreover, plant tissues are normally thick (having multiple cell layers), and can have strong autofluorescence, especially in green leaves. Therefore, optimized culturing and imaging conditions are essential for successful live cell studies in plants. In this protocol, specific chromatin loci (centromeres) in Arabidopsis are tagged with centromere-specific histone 3 variant (HTR12)-fluorescent protein (FP) fusions, and transgenic plants expressing these fusions are generated. Three- and four-dimensional imaging techniques are used to visualize the organization and dynamics of these specific chromatin loci in interphase and through mitosis. The procedure can be modified easily to accommodate other proteins or structures of interest.