A confocal technique applicable to studies of cellular pH-related signaling in plants

pH may act as a crucial signal in both animal and plant cells. It is very difficult to monitor pH signals and this has largely hindered progress in the investigation of pH signaling, particularly systematic pH signaling. Here, we report the development of a confocal technique to monitor leaf apoplastic pH in intact plants, which is particularly suitable for the studies on root to shoot signaling. A variety of different pH indicators and plant species were tested. It was found that different pH indicators, for example, 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresce (BCECF), SNARF-4F 5-(and-6)-carboxylic acid (SNARF) and DM-NERF (NERF), were of different properties, and to successfully monitor pH at a sub-cellular level, the comparability between the pH indicator and plant species must be involved according to their suitable pH range and loading characteristics. The loading characteristics of different pH indicators differ with different plant species, cell types and their developing stages. No matter what methods were adopted, BCECF and SNARF could not be loaded specifically in the leaf apoplast in sunflower, tomato, and Comelina communis L. In contrast, regardless of the methods adopted, NERF could be loaded efficiently and specifically in the leaf apoplast in C. communis, but not in other plants. In C. communis, the determination coefficient for in vitro and in situ calibration of NERF was very high, which was respectively 0.9951 and 0.9916, and therefore, the adoption of NERF together with C. communis could construct an ideal experimental system that is suitable for the investigation of pH systematic signaling. Ratio image analysis demonstrated that the leaf apoplastic pH was about 5.5 in non-stressed conditions, and water deficit could trigger an increase in pH by about half a pH unit, which is the first evidence to directly indicate that pH is able to act as a systematic signal under water deficit conditions.