Multiple feedbacks on self-organized morphogenesis during plant regeneration.

Decades of research have primarily emphasized genetic blueprint as the driving force behind plant regeneration. The flow of information from genetics, which manifests as biochemical properties, including hormones, has been extensively implicated in plant regeneration. However, recent advancements have unveiled additional intrinsic modules within this information flow. Here, we explore the three core modules of plant regeneration: biochemical properties, mechanical forces acting on cells, and cell geometry. We debate their roles and interactions during morphogenesis, emphasizing the potential for multiple feedbacks between these core modules to drive pattern formation during regeneration. We propose that de novo organ regeneration is a self-organized event driven by multidirectional information flow between these core modules.

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.

Age, Wound Size, and Position of Injury - Dependent Vascular Regeneration Assay in Growing Leaves.

Recurring damage to the aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assays for aerial plant parts such as the stem and inflorescence stalk are well established, those for leaf vasculature remain unexplored. Recently, we established a new vascular regeneration assay in growing leaves and discovered the underlying molecular mechanism. Here, we describe the detailed stepwise method for the incision and regeneration assay used to study leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy, and other experimental approaches, we further show that the age of the leaf as well as the position and size of the injury determine the overall success rate of regeneration. This easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. The assay can be readily combined with cellular and molecular biology techniques.

Model systems for regeneration: Arabidopsis.

Plants encompass unparalleled multi-scale regenerative potential. Despite lacking specialized cells that are recruited to injured sites, and despite their cells being encased in rigid cell walls, plants exhibit a variety of regenerative responses ranging from the regeneration of specific cell types, tissues and organs, to the rebuilding of an entire organism. Over the years, extensive studies on embryo, shoot and root development in the model plant species Arabidopsis thaliana have provided insights into the mechanisms underlying plant regeneration. These studies highlight how Arabidopsis, with its wide array of refined molecular, genetic and cell biological tools, provides a perfect model to interrogate the cellular and molecular mechanisms of reprogramming during regeneration.

Regrowing the damaged or lost body parts.

Plants display extraordinary ability to revive tissues and organs lost or damaged in injury. This is evident from the root tip restoration and classical experiments in stem demonstrating re-establishment of vascular continuity. While recent studies have begun to unravel the mechanistic understanding of tissue restoration in response to injury in underground plant organs, the molecular mechanisms of the same in aerial organs remain to be ventured deeper. Here, we discuss the possibility of unearthing the regulatory mechanism that can confer universal regeneration potential to plant body and further provide a comprehensive understanding of how tissue and organ regeneration gets triggered in response to mechanical injury and later gets terminated after re-patterning and regaining the appropriate size.