Background

Plants perform photosynthesis during the day to convert light energy into usable chemical energy. Many plant species store this energy as polysaccharides by accumulating starch granules within chloroplasts. At night, starch granules are broken down into soluble sugars, which are then distributed to sink organs for use in growth and development [1,2]. The products mobilized from starch degradation also mediate osmotic stress tolerance [3] and facilitate stomatal opening in guard cells [4]; thus, the biosynthesis and degradation of chloroplast starch granules are crucial processes in plants.

Several staining methods have been developed for observing chloroplast starch granules in leaves, enabling the quantification and investigation of their morphology. Conventional staining methods such as iodine, toluidine blue, and periodic acid Schiff staining have been widely used [5–7]; however, these staining methods involve multiple procedures and chemical fixation, preventing rapid and/or live-cell imaging. Chloroplast starch granules have also been visualized using fluorescent staining methods, including modified pseudo-Schiff propidium iodide [4,8] and safranin O staining [9], but these methods generate non-specific signals. It is therefore necessary to develop a simple staining method that enables the observation of living cells and shows good specificity for chloroplast starch granules.

To address this need, we recently established a fluorescein staining method for visualizing chloroplast starch granules fluorescently in various living plant cells [10]. Fluorescein staining only requires the submergence of leaf tissue in staining solution for 10 min. Due to its high specificity for chloroplast starch granules, fluorescent images of stained leaf tissue can be used to quantify the cellular starch content. Fluorescein staining is therefore a valuable analytical tool for understanding chloroplast starch granules in various plant species.