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.

Faculty of Science Department of Experimental Plant Biology Charles University Viničná 5 CZ 128 43 Prague Czechia

Max Planck Institute for Plant Breeding Research Carl Von Linné Weg 10 50829 Cologne Germany

Zobrazit více v PubMed

Kaiser S, Scheuring D. To lead or to follow: contribution of the plant vacuole to cell growth. Front Plant Sci. 2020;11:553. 10.3389/fpls.2020.00553. PubMed DOI PMC

Cui Y, Cao W, He Y, Zhao Q, Wakazaki M, Zhuang X, et al. A whole-cell electron tomography model of vacuole biogenesis in PubMed DOI

Scheuring D, Minina EA, Krueger F, Lupanga U, Krebs M, Schumacher K. Light at the end of the tunnel: FRAP assays combined with super resolution microscopy confirm the presence of a tubular vacuole network in meristematic plant cells. Plant Cell. 2024;36:4683–91. 10.1093/plcell/koae243. PubMed DOI PMC

Oda Y, Higaki T, Hasezawa S, Kutsuna N. New insights into plant vacuolar structure and dynamics. Int Rev Cell Mol Biol. 2009;277:103–35. 10.1016/S1937-6448(09)77003-0. PubMed DOI

Verbelen JP, De Cnodder T, Le J, Vissenberg K, Baluska F. The root apex of PubMed DOI PMC

Löfke C, Dünser K, Scheuring D, Kleine-Vehn J. Auxin regulates SNARE-dependent vacuolar morphology restricting cell size. Elife. 2015;4:e05868. 10.7554/eLife.05868. PubMed DOI PMC

Scheuring D, Löfke C, Krüger F, Kittelmann M, Eisa A, Hughes L, et al. Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression. Proc Natl Acad Sci U S A. 2016;113:452–7. 10.1073/pnas.1517445113. PubMed DOI PMC

Bibikova T, Gilroy S. Root hair development. J Plant Growth Regul. 2002;21:383–415. 10.1007/s00344-003-0007-x. DOI

Dünser K, Gupta S, Herger A, Feraru MI, Ringli C, Kleine-Vehn J. Extracellular matrix sensing by FERONIA and Leucine-Rich Repeat Extensins controls vacuolar expansion during cellular elongation in PubMed DOI PMC

Osorio-Navarro C, Neira-Valenzuela G, Sierra P, Adamowski M, Toledo J, Norambuena L. The configuration of the vacuole is driven by clathrin-mediated trafficking in root cells of PubMed DOI

Krüger F, Schumacher K. Pumping up the volume - vacuole biogenesis in PubMed DOI

Rojo E, Gillmor CS, Kovaleva V, Somerville CR, Raikhel NV. VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis. Dev Cell. 2001;1:303–10. 10.1016/s1534-5807(01)00024-7. PubMed DOI

Gattolin S, Sorieul M, Hunter PR, Khonsari RH, Frigerio L. In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots. BMC Plant Biol. 2009;9:133. 10.1186/1471-2229-9-133. PubMed DOI PMC

Segami S, Makino S, Miyake A, Asaoka M, Maeshima M. Dynamics of vacuoles and H+-pyrophosphatase visualized by monomeric green fluorescent protein in PubMed DOI PMC

Kaiser S, Eisa A, Kleine-Vehn J, Scheuring D. NET4 modulates the compactness of vacuoles in PubMed DOI PMC

Julian J, Gao P, Del Chiaro A, Carlos De La Concepcion J, Armengot L, Somssich M, et al. Atg8ylation of vacuolar membrane protects plants against cell wall damage. Nat Plants. 2025;11:321–39. 10.1038/s41477-025-01907-z. PubMed DOI PMC

Müller J, König Y, Kaiser S, Löfke C, Krebs M, Scheuring D. Salicylic acid restricts cell elongation and induces changes of vacuolar morphology and pH. bioRxiv. 2024. 10.1101/2024.09.06.611645. DOI

Tiernan WK, Castro GV, Shipman K, Rodriguez-Furlan C. Protocol to fluorescently stain vacuoles in PubMed DOI PMC

Kollárová E, Baquero Forero A, Yildiz AB, Kočová H, Žárský V, Cvrčková F. The DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. 10.1038/nmeth.2019. PubMed DOI PMC

Galway ME, Heckman JW Jr, Schiefelbein JW. Growth and ultrastructure of Arabidopsis root hairs: the rhd3 mutation alters vacuole enlargement and tip growth. Planta. 1997;201:209–18. 10.1007/BF01007706. PubMed DOI

Virtanen P, Gommers R, Oliphant TE, Haberland M, Reddy T, Cournapeau D, et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods. 2020;17(3):261–72. 10.1038/s41592-019-0686-2. PubMed DOI PMC

Hunter JD. Matplotlib: a 2D graphics environment. Comp Sci Eng. 2007;9:90–5. 10.1109/MCSE.2007.55. DOI

McKinney W. Data structures for statistical computing in Python. In SciPy Proceedings; 2010: 56–61. 10.25080/Majora-92bf1922-00a.

The pandas development team. pandas-dev/pandas: Pandas. 2023. Zenodo. 10.5281/zenodo.10304236. DOI

R core team. R: a language and environment for statistical computing. https://www.R-project.org. Accessed on 31 July 2025.

Wickham H, Averick M, Bryan J, Chang W, D’Agostino McGowan L, François R, et al. Welcome to the Tidyverse. J Open Source Softw. 2019;4:1686. 10.21105/joss.01686. DOI

Spitzer M, Wildenhain J, Rappsilber J, Tyers M. BoxPlotR: a web tool for generation of box plots. Nat Methods. 2014;11:121–2. 10.1038/nmeth.2811. PubMed DOI PMC

Waskom ML. Seaborn: statistical data visualization. J Open Source Softw. 2021;6:3021. 10.21105/joss.03021. DOI

Shapiro SS, Wilk MB. An analysis of variance test for normality (complete samples). Biometrika. 1965;52:591–611. 10.1093/biomet/52.3-4.591. DOI

Mann HB, Whitney DR. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat. 1947;18:50–60. 10.1214/aoms/1177730491. DOI

Student. The probable error of a mean. Biometrika. 1908;6:1–25. 10.2307/2331554. DOI

Olejnik S, Algina J. Generalized eta and omega squared statistics: measures of effect size for some common research designs. Psychol Methods. 2003;8:434–47. 10.1037/1082-989X.8.4.434. PubMed DOI

Schober P, Boer C, Schwarte LA. Correlation coefficients: appropriate use and interpretation. Anesth Analg. 2018;126:1763–8. 10.1213/ANE.0000000000002864. PubMed DOI

Cao W, Li Z, Huang S, Shi Y, Zhu Y, Lai MN, et al. Correlation of vacuole morphology with stomatal lineage development by whole-cell electron tomography. Plant Physiol. 2022;188:2085–100. 10.1093/plphys/kiac028. PubMed DOI PMC

Kutsuna N, Kumagai F, Sato MH, Hasezawa S. Three-dimensional reconstruction of tubular structure of vacuolar membrane throughout mitosis in living tobacco cells. Plant Cell Physiol. 2003;44:1045–54. 10.1093/pcp/pcg124. PubMed DOI

Mirasole FM, Nastasi SP, Cubero-Font P, De Angeli A. Vacuolar control of stomatal opening revealed by 3D imaging of the guard cells. Sci Rep. 2023;13:7647. 10.1038/s41598-023-34273-x. PubMed DOI PMC

Barbier de Reuille P, Routier-Kierzkowska AL, Kierzkowski D, Bassel GW, Schüpbach T, Tauriello G, et al. MorphoGraphX: a platform for quantifying morphogenesis in 4D. Elife. 2015;4:05864. 10.7554/eLife.05864. PubMed DOI PMC

Kaiser S, Eisele S, Scheuring D. Vacuolar occupancy is crucial for cell elongation and growth regardless of the underlying mechanism. Plant Signal Behav. 2021;16:1922796. 10.1080/15592324.2021.1922796. PubMed DOI PMC

Dünser K, Schöller M, Rößling AK, Löfke C, Xiao N, Pařízková B, et al. Endocytic trafficking promotes vacuolar enlargements for fast cell expansion rates in plants. Elife. 2022;11:e75945. 10.7554/eLife.75945. PubMed DOI PMC

Yamagami A, Chieko S, Sakuta M, Shinozaki K, Osada H, Nakano A, et al. Brassinosteroids regulate vacuolar morphology in root meristem cells of PubMed DOI PMC

Dragwidge JM, Scholl S, Schumacher K, Gendall AR. NHX-type Na+(K+)/H+ antiporters are required for TGN/EE trafficking and endosomal ion homeostasis in PubMed DOI

De Vriese K, Himschoot E, Dünser K, Nguyen L, Drozdzecki A, Costa A, et al. Identification of novel inhibitors of auxin-induced Ca PubMed DOI PMC

Fagerland MW. T-tests, non-parametric tests, and large studies–a paradox of statistical practice? BMC Med Res Methodol. 2012;12:78. 10.1186/1471-2288-12-78. PubMed DOI PMC

Poncet A, Courvoisier D, Combescure C, Perneger TV. Normality and sample size do not matter for the selection of an appropriate statistical test for two-group comparisons. Methodology. 2016;12:61–71. 10.1027/1614-2241/a000110. DOI

Kočová H, Žárský V, Cvrčková F. The fate and function of the Arabidopsis Class I formin AtFH1 during central vacuole biogenesis in the rhizodermis. Front Plant Sci. 2025;16:1685260. 10.3389/fpls.2025.1685260. PubMed DOI PMC