The Tonoplast Topology Index-a new metric for describing vacuole organization.

BACKGROUND: The plant vacuole arises by orchestrated interplay of membrane trafficking, cytoskeletal rearrangements and a variety of signaling pathways. In the root, the characteristic large central vacuole develops by endomembrane reorganization occurring mainly in the transition zone. The vacuole's bounding membrane-the tonoplast-can be visualized in vivo using fluorescent protein markers, allowing for quantitative analysis of confocal microscopy images. Tonoplast organization can thus serve as a sensitive indicator of changes to any of the processes involved in vacuole biogenesis. The Vacuolar Morphology Index (VMI) is widely accepted as a quantitative measure of vacuole structure. However, this metric has two drawbacks-it only reflects the size of the largest vacuolar compartment (missing therefore possible differences in the organization of smaller compartments), and its determination is labor intensive, limiting its use on large datasets. RESULTS: We developed an alternative metric for describing vacuole organization, named the Tonoplast Topology Index (TTI), which overcomes the above-mentioned shortcomings of the VMI. We compared the performance of our protocol with VMI on a simulated dataset and on real data. To validate the methods´ performance, we used it to confirm the previously reported differences in vacuole shape and size between Arabidopsis thaliana roots grown on the surface of an agar medium compared to those embedded inside the agar. Both VMI and TTI could efficiently detect the relatively subtle changes in vacuole organization depending on the position of the root in the agar, and provided correlated results. However, only TTI produced data with close to normal value distribution, simplifying subsequent statistical evaluation. CONCLUSIONS: We present the protocol for TTI determination as a two-stage semi-automated procedure involving microscopic image analysis employing an ImageJ macro and subsequent processing of numeric data in the Jupyter Notebook environment, together with benchmarking image data. Since this implementation is freeware-based, platform-independent and (relatively) user-friendly, we hope it will find its use as a high throughput, added value alternative to the VMI metric.