Quantitative Phenotyping of Ion Fluxes in Tip-Growing Cells Under Varying Growth Regimes: A Data Analysis Protocol.

Tip-growing cells exhibit complex growth regimes in vitro, alternating between growing and non-growing intervals, oscillatory or more steady behavior. In Arabidopsis thaliana, pollen tube growth arrest is often accompanied by spiking behavior in intracellular ion concentrations and extracellular ion fluxes. Thus, selecting comparable growing regimes is critical for quantifying ion dynamics across cells and genotypes. Defining non-growing regimes is a fundamental step to filter out their associated data points. Here, we provide computational and statistical procedures for the quantitative phenotyping of ion fluxes associated with growth dynamics in tip-growing cells. The goal is to provide reliable estimates of ion fluxes given pairwise time-series comparisons (ion fluxes vs. growth rate), focused on growth-associated intervals and avoiding data stemming from regimes associated with growth arrest. We consider extracellular ion fluxes in growing tubes, but the analysis is applicable to other quantitative variables and tip-growing cells. After visualizing both series in a common timeframe, we extract the growth rate baseline and determine the non-growing regime threshold with a Gaussian Mixture Model, then predict the growth state at sampled flux times, filtering, and finally quantification. The protocol is presented in R but is of general use, since multiple software routines can yield similar results.