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Evergreen species of temperate zone acclimate to seasonal climates by reorganizations of mesophyll cell structure including chloroplast movement as a photoprotective reaction. However the exact factor inducing structural changes is still unexplored. To reveal the specific pattern of chloroplast arrangement during the annual cycle and the effect of temperature on their movement, the mesophyll cell structure in Pinus sylvestris grown out- and indoors was studied. The serial block-face scanning electron microscopy (SBF SEM) was used for the 3D imaging of mesophyll cells to show the spatial position and shape modification of chloroplasts. It has been shown that during the growing season, chloroplasts have a well-developed thylakoid system, they are located along the cell wall and occupy predominantly the part of the cell wall faced the intercellular airspace. Chloroplast movement starts in October-November, and during the winter they aggregate in the cell lobes clumping together. At that time, the thylakoid system is reorganised and consists mainly of long doubled thylakoids and small grana. The 3D reconstruction shows that the chloroplasts are irregularly oriented, swollen, and develop multiple protrusions filled by stroma that can be recognized as stromules. In indoor plants, seasonal reorganization of the mesophyll ultrastructure does not occur suggesting low temperatures but not photoperiod and light quality induce seasonal chloroplast movement in P. sylvestris mesophyll. Finally, we indicate 3D reconstruction is a powerful tool in study of low temperature-induced change of chloroplast positioning.