Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

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.

Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes

“…In a previous study using isolated chloroplasts, RhB and NR staining were used to distinguish between intact and disrupted chloroplasts; only intact chloroplasts were visualized as spherical shapes using the two fluorescent dyes, indicating that RhB and NR possibly stain the OEM and/or IEM of isolated chloroplasts (An et al, 2021 ). RhB and NR have also been reported to be membrane‐permeable dyes (Greenspan et al, 1985 ; Grolig & Wagner, 1987 ; Halim & Webley, 2015 ; Mottram et al, 2012 ).…”

A novel in-situ-process technique constructs whole circular cpDNA library

“…Among the developed cpDNA extraction techniques with an aqueous phase (like the CTBA method and alkaline lysis) caused the fragmentation of the circular cpDNA and a few residual non-target DNA impurities are inevitable, the high-quality circular cpDNA is not perfect in the conventional extraction method. The method in the choiceness article of cp genome extraction between 1963 and 2021 is chronicled in Additional file 2 : Table S1[ 3 – 6 , 10 , 14 , 16 – 49 ]. Updating the techniques in the cp extraction method is the consequence of the consistent efforts of the industrious scientific research people, although the space is constrained to be outlined.…”