Bundle sheath cell-dependent chloroplast movement in mesophyll cells of C ₄ plants analyzed using live leaf-section imaging

In C ₄ plants, mesophyll (M) chloroplasts aggregate toward bundle sheath (BS) cells in response to environmental stress, which would contribute to C ₄ photosynthetic cycle adjustment between M and BS cells. However, it remains unclear whether mesophyll chloroplast movement is an intercellular response mediated by BS cells. One major challenge to resolving this is the difficulty in observing how M chloroplasts aggregate toward adjacent BS cells due to scattering and absorption of observation light in live-leaf tissues. We established a live leaf-section imaging technique that enables the long-term observation of sections of chemically unfixed leaf blades, with which we quantitatively analyzed M chloroplast movements. Another challenge in clarifying the contribution of BS cells to M chloroplast movement is the selective ablation of BS cells without impairing their function of M cells. To investigate the necessity of BS cells for M chloroplast movement, we developed a method to remove BS cells only based on differences in shape and size between M and BS cells. We also found that chloroplasts in M cells without adjacent BS cell contents did not show typical aggregative movement but showed a light-avoidance response. This indicates that the M chloroplast aggregative movement occurs during communication with BS cells.