A method for characterizing phenotypic changes in highly variable cell populations and its application to high content screening of Arabidopsis thaliana protoplasts

Abstract

Quantitative image analysis procedures are necessary for the automated discovery of effects of drug treatment in large collections of fluorescent micrographs. When compared to their mammalian counterparts, the effects of drug conditions on protein localization in plant species are poorly understood and underexplored. To investigate this relationship, we generated a large collection of images of single plant cells after various drug treatments. For this, protoplasts were isolated from six transgenic lines of A. thaliana expressing fluorescently tagged proteins. Nine drugs at three concentrations were applied to protoplast cultures followed by automated image acquisition. For image analysis, we developed a cell segmentation protocol for detecting drug effects using a Hough-transform based region of interest detector and a novel cross-channel texture feature descriptor. In order to determine treatment effects, we summarized differences between treated and untreated experiments with an L1 Cramér-von Mises statistic. The distribution of these statistics across all pairs of treated and untreated replicates was compared to the variation within control replicates to determine the statistical significance of observed effects. Using this pipeline, we report the dose dependent drug effects in the first high-content Arabidopsis thaliana drug screen of its kind. These results can function as a baseline for comparison to other protein organization modeling approaches in plant cells.

Determination of protoplast growth properties using quantitative single-cell tracking analysis

“…Our approach is based on the fact that quantitative microscopic analysis of single-cell responses is well established and widely used for animal and yeast cells while corresponding applications on plant single cells is largely restricted only to the very early time points occurring within minutes to a few hours after isolation [ 6 , 19 , 33 , 52 ]. However, monitoring of growth properties over time, several days after isolation until the initiation of proliferation, has only been rarely done so far [ 35 ].…”

Handling Complexity in Animal and Plant Science Research—From Single to Functional Traits: Are We There Yet?

“…With the expected and rapid increases in knowledge in agriculture research, it is imperative that methodologies for defining phenotypes are clear and standardized [ 11 ]. Furthermore, the detection of any mutations or altered expressions of genes depends on phenotypic screening methods and the ability to detect variations from normal [ 12 ]. The next challenge will be to develop fast, efficient, systematic and comprehensive phenotypic screening procedures and tools that will permit comparison among laboratories [ 12 ].…”

“…Furthermore, the detection of any mutations or altered expressions of genes depends on phenotypic screening methods and the ability to detect variations from normal [ 12 ]. The next challenge will be to develop fast, efficient, systematic and comprehensive phenotypic screening procedures and tools that will permit comparison among laboratories [ 12 ]. It is well established that molecular breeding based on DNA markers has led to advances in breeding; however, not in the way that it was anticipated initially [ 13 ].…”