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

Abstract

Background Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions. Results High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4). Conclusions AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

Multiscale chromatin dynamics and high entropy in plant iPSC ancestors

“…We aimed at high-throughput imaging of plant iPSC ancestors in culture, similar to what had been undertaken previously for analysing cellular morphology ( Dawson et al, 2022 ), but focusing here on chromatin markers. For this, we generated a dual reporter Arabidopsis line expressing both the H1.2–GFP and H2B–RFP markers (selected following the strategy explained above), and used it to establish and benchmark the growth conditions, imaging setup and an image analysis pipeline.…”

Agrobacterium-Mediated Transformation of the Dwarf Soybean MiniMax

“…Renilla (Rluc) luciferase acts as a gene of interest and is expressed by the double enhanced cauliflower mosaic promoter db35S. Rluc is used to determine the level of gene expression based on the genomic location of T-DNA integration [24]. T-DNA sequence available upon request.…”

Multiscale chromatin dynamics and high entropy in plant iPSC ancestors

“…We aimed at high-throughput imaging of plant iPSC ancestors in culture, similar than done previously for analysing cellular morphology (Dawson et al, 2022), but focusing here on chromatin markers. For this, we generated a dual reporter Arabidopsis line expressing both the H1.2-GFP and H2B-RFP markers (selected following the strategy explained above), and used it to establish and benchmark the growth conditions, imaging setup and an image analysis pipeline.…”