Protocol for real-time imaging, polar protein quantification, and targeted laser ablation of regenerating shoot progenitors in Arabidopsis.

Here, we provide a protocol for real-time tracking of regenerating shoot progenitors, combined with polar protein quantification and targeted laser ablation of callus cells in Arabidopsis. Using Arabidopsis strains expressing GFP-labeled polar auxin efflux carrier, PINFORMED 1 (PIN1) protein, we detail steps to prepare the callus for time-lapse confocal imaging and track the progenitors expressing PIN1-GFP, followed by mapping and quantifying PIN1 polarity using Fiji/ImageJ. We then describe targeted laser ablation of cells and subsequent time-lapse imaging to study regeneration. For complete details on the use and execution of this protocol, please refer to Varapparambath et al. (2022).1.

Mechanical conflict caused by a cell-wall-loosening enzyme activates de novo shoot regeneration.

Cellular heterogeneity is a hallmark of multicellular organisms. During shoot regeneration from undifferentiated callus, only a select few cells, called progenitors, develop into shoot. How these cells are selected and what governs their subsequent progression to a patterned organ system is unknown. Using Arabidopsis thaliana, we show that it is not just the abundance of stem cell regulators but rather the localization pattern of polarity proteins that predicts the progenitor's fate. A shoot-promoting factor, CUC2, activated the expression of the cell-wall-loosening enzyme, XTH9, solely in a shell of cells surrounding the progenitor, causing different mechanical stresses in these cells. This mechanical conflict then activates cell polarity in progenitors to promote meristem formation. Interestingly, genetic or physical perturbations to cells surrounding the progenitor impaired the progenitor and vice versa. These suggest a feedback loop between progenitors and their neighbors for shoot regeneration in the absence of tissue-patterning cues.