A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H⁺-ATPases and V-type H⁺-PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.

Significance: Terpenoid indole alkaloids are known to be valuable bioactive compounds. In situ RNA hybridization of gene expression of the terpenoid indole alkaloid (TIA) synthetic enzymes has suggested that the TIA metabolic pathway in Catharanthus roseus stem tissue involves the successive metabolic flow of four types of cells: internal phloem-associated parenchyma, epidermal, idioblast, and laticifer cells. It has never been directly determined in which of these cells these TIA intermediates are localized. The present study showed, using both Imaging mass spectrometry (MS) and Single-cell MS, that many kinds of TIA intermediates, including catharanthine and serpentine, were accumulated in idioblast and laticifer cells. The developed methods should prove useful for studying other aspects of secondary metabolism in plants.

The intracellular membrane dynamics of Arabidopsis cells under high salt treatment were investigated. When Arabidopsis was treated with high levels of NaCl in hydroponic culture, root tip cells showed rapid changes in the vacuolar volume, a decrease in the number of small acid compartments, active movement of vesicles and accumulation of Na⁺ both in the central vacuole and in the vesicles around the main vacuole observed with the Na⁺-dependent fluorescence of Sodium Green. Detailed observation of Arabidopsis suspension-cultured cells under high salt treatment showed a similar pattern of response to that observed in root tip cells. Immunostaining of suspension-cultured cells with antibodies against AtNHX1 clearly showed the occurrence of dotted fluorescence in the cytoplasm only under salt treatment. We also confirmed the existence of AtNHX1 in the vacuolar membrane isolated from suspension-cultured cells with immunofluorescence. Knockout of the vacuolar Q_a-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein VAM3/SYP22 caused an increase in salt tolerance. In mutant plants, the distribution of Na⁺ between roots and shoots differed from that of wild-type plants, with Na⁺ accumulating more in roots and less in the shoots of the mutant plants. The role of vesicle traffic under salt stress is discussed.

We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP₆) synthesis in suspension-cultured cells of Catharanthus. InsP₆ and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP₆ was less than 50 nm, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP₆ was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K⁺, Ca²⁺, or Zn²⁺. However, there was a strong tendency for InsP₆ to accumulate in the vacuole in the presence of Ca²⁺ and in nonvacuolar compartments when supplied with Zn²⁺, possibly due to precipitation of InsP₆ with Zn²⁺ in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP₆ accumulation. The amounts of both Ins(3)P₁ myo-inositol monophosphate synthase, a key enzyme for InsP₆ synthesis, and Ins(1,4,5)P₃ kinase were unrelated to the level of accumulation of InsP₆. The mechanisms for InsP₆ synthesis and localization into vacuoles in plant cells are discussed.

The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics.