Biolistic delivery of Ca²⁺ dyes into plant and algal cells

Summary

In eukaryotes, changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) are associated with a number of environmental and developmental stimuli. However, measuring [Ca²⁺]_cyt changes in single plant or algal cells is often problematic. Although a wide range of Ca²⁺-sensitive fluorescent dyes is available, they are often difficult to introduce into plant cells. Micro-injection is the most robust method for dye loading, but is time-consuming, technically demanding, and unsuitable in many cell types. To overcome these problems, we have adapted biolistic techniques to load Ca²⁺-sensitive dyes into guard cells of the flowering plant, Commelina communis, cells of the green alga Chlamydomonas reinhardtii, and zygotes of the brown alga, Fucus serratus. Using this approach, we have been able to monitor [Ca²⁺]_cyt changes in response to various stimuli, including a novel [Ca²⁺]_cyt response in C. reinhardtii. The method allows the use of free acid and dextran-conjugated dyes. Biolistic loading of differentiated plant cells is easier, quicker, and more widely applicable than micro-injection, and should broaden the study of plant signal transduction.