Abstract
Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.
Highlights
Chloroplasts are plant-specific organelles encapsulated by double membranes, inside which contain thylakoids, stroma, and other structures
We found that isolated chloroplasts from the middle layer of Percoll gradient are not totally intact and this can be quickly observed with a fluorescence microscope
Obtaining intact chloroplasts is important for the study of chloroplasts
Summary
Chloroplasts are plant-specific organelles encapsulated by double membranes, inside which contain thylakoids, stroma, and other structures. Chloroplasts are the major site of photosynthesis They are involved in many other important biochemical processes in plants, such as the biosynthesis of fatty acids, amino acids, and plant hormones (Azevedo et al, 2006; Lopez-Juez, 2007; Schaller and Stintzi, 2009; Sendon et al, 2011; Mano and Nemoto, 2012; Wang and Benning, 2012). Biochemical, physiological, and proteomic analyses are common methods to study the functions and development of chloroplasts (Gao et al, 2003, 2006; Baginsky and Gruissem, 2004; Block et al, 2007; Lopez-Juez, 2007; Su and Lai, 2017; Fuertauer et al, 2019). Due to the difference of the densities between broken chloroplasts and intact
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