Using a Live Analysis System to Study Amyloplast Replication in Arabidopsis Ovule Integuments

Amyloplasts, non-photosynthetic plastids specialized for starch synthesis and storage, proliferate in storage tissue cells of plants. To date, studies of amyloplast replication in roots and the ovule nucelli from various plant species have been performed using electron and fluorescence microscopy. However, a complete understanding of amyloplast replication remains unclear due to the absence of experimental systems capable of tracking their morphology and behavior in living cells. Recently, we demonstrated that Arabidopsis ovule integument could provide a platform for live-cell imaging of amyloplast replication. This system enables precise analysis of amyloplast number and shape, including the behavior of stroma-filled tubules (stromules), during proplastid-to-amyloplast development in post-mitotic cells. Here, we provide technical guidelines for observing and quantifying amyloplasts using conventional fluorescence microscopy in wild-type and several plastid-division mutants of Arabidopsis. Key features • Novel approach for investigating amyloplast differentiation and replication in plant cells. • Detection of stroma-labeled amyloplasts in whole-mount ovules using conventional fluorescence microscopy. • Facilitates quantitative and comparative analysis of amyloplast proliferation using various Arabidopsis resources. • Enables high-resolution analysis of changing amyloplast and stromule morphologies in living cells.