Background

Chloroplasts are organelles responsible for photosynthesis in leaves that change their intracellular positions in response to various environmental stimuli. In C₄ plants, which perform photosynthesis across two types of photosynthetic cells, mesophyll and bundle sheath cells, an aggregative movement of mesophyll chloroplasts has been observed. Under stress conditions such as high-intensity light, drought, and salinity, mesophyll chloroplasts aggregate to bundle sheath cells [1–3]. Techniques such as transmittance measurements, band assays, polarized light irradiation, and microbeam irradiation have been used to elucidate the mechanisms involved in chloroplast movement [4–6]. However, these conventional methods have been insufficient for investigating the mechanism of mesophyll chloroplast aggregative movement in C₄ plants, particularly the potential involvement of adjacent bundle sheath cells. This is because C₄ plants possess a concentric leaf structure composed of multiple cell layers, and mesophyll chloroplasts aggregate toward the inner bundle sheath cells. Such inward movements cannot be accurately measured using transmittance-based methods or similar techniques, and the behavior of individual mesophyll chloroplasts relative to adjacent bundle sheath cells within the tissue remains unclear. Although thin sectioning after chemical fixation allows for the precise observation of chloroplast positioning, biological activity ceases after fixation, preventing the assessment of physiological responses.

To address these challenges, we developed a live leaf-section imaging technique that enables the long-term observation of chloroplast movement in unfixed leaf sections. This technique requires a certain level of technical proficiency; however, it is fundamentally simple because it involves thin sectioning live tissues without chemical fixation. This allows for quantitative analysis of chloroplast movement relative to bundle sheath cells, providing insights into intercellular interactions and movement dynamics. Furthermore, by adjusting the sectioning angle and thickness of the unfixed leaf sections, we successfully prepared sections that excluded the contents of bundle sheath cells, enabling investigation of the role of adjacent bundle sheath cells in chloroplast movement. Using these approaches, we demonstrated that mesophyll chloroplast movement in C₄ plants is an intercellular response [7].

In this protocol, using the C₄ plant Eleusine coracana (finger millet) as an example, we describe the preparation of leaf sections, the setup for observation, and the analytical methods used to track chloroplast movement. Additionally, we introduce a hand-sectioning method that allows for the preparation of unfixed sections in plant species that are difficult to section using a vibrating microtome owing to leaf hardness. Our method enables the observation of entire leaf tissues in a living state, making it applicable to the study of chloroplast movement and providing a valuable tool for analyzing tissue-level physiological responses.