Background

Plastids are double membrane–bound organelles found in the cells of seed plants that exhibit unique features of functional and morphological differentiation. These include chloroplasts in leaves, chromoplasts in flowers and fruits, and leucoplasts in general non-photosynthetic tissues [1]. Amyloplasts are non-photosynthetic plastids specialized for starch synthesis and storage. Amyloplasts develop from undifferentiated proplastids in meristematic tissues and are distributed in storage tissues such as roots, stems, and seeds, as well as in gravity-sensing tissues (for example, the stem endodermis and root cap columella) [2]. As starch is an important source of nutrients in human diets, the differentiation and proliferation of amyloplasts have long been an important area of crop biology research.

Plastids possess their DNA and typically replicate by binary fission [3,4]. In leaf mesophyll cells of the model plant Arabidopsis thaliana, chloroplast division is initiated by the formation of the stromal FtsZ ring and involves approximately 20 nuclear-encoded gene products located in the stroma, inner-envelope membrane, outer-envelope membrane, or cytoplasm [5,6]. In contrast, the mechanisms governing amyloplast proliferation remain largely unknown, partly due to the lack of an experimental system to track their number and behavior in specific cells.

We recently proposed that the Arabidopsis ovule integument could provide a platform for live-cell imaging of amyloplast replication and morphology, including the dynamics of stroma-filled tubules (stromules) [7]. This method may open a way to analyzing the function and regulation of plastid-division proteins during amyloplast proliferation in non-photosynthetic cells. In this protocol, we provide technical tips for conventional fluorescence microscopy of integument amyloplasts using wild-type (WT) and plastid-division mutants of Arabidopsis.