Intracellular positioning of mesophyll chloroplasts following to aggregative movement in Setaria viridis analysed three-dimensionally with a confocal laser scanning microscope

Abstract

Chloroplasts can change their intracellular position responding to environmental conditions. In addition to the well-known photorelocation movements, i.e., accumulation and avoidance movements in response to light, mesophyll chloroplasts in C4 plants change their intracellular positioning from the cell periphery to the bundle sheath cell side (aggregative movement) in response to environmental stresses such as drought, salinity, and hyperosmosis, under light. Previous studies on the aggregative movement have been limited in two-dimensional (2D) information on the transverse sections of leaves, because aggregated mesophyll chloroplasts gather inside the leaf tissues, which need to be cut for observation. However, the 2D analysis on the cross sections is difficult to investigate accurately the aggregated chloroplasts overlapped each other in the depth direction. Therefore, there are few studies examining the anatomical features of the aggregated chloroplasts at the cellular level. Here in this study, we established the workflow for three-dimensional (3D) observation using a confocal laser scanning microscope (CLSM), which can investigate a thick section as a stack of optical sections, followed by 3D reconstruction of mesophyll cells and chloroplasts. Using this method, we visualized the 3D representations of mesophyll cells of green foxtail (Setaria viridis), which is a model of C4 plant, and investigated the chloroplasts individually and quantified their structures or intracellular positions before and after the aggregative movement. The 3D data of individual chloroplasts in a whole cell revealed that the aggregated positioning is independent with chloroplast volume or surface area, and that chloroplasts did not change their shape before and after the movement.