Isolation of Intact Vacuoles from Root Protoplasts and Elemental Analysis.

The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins. Graphical abstract.