Reconstruction of the 3D structure and developmental history of plant cells and tissues

TL;DR

This paper introduces protocols for 3D reconstruction of plant cells and tissues from microscopy images, enabling detailed lineage tracing and subcellular analysis across various plant materials.

Contribution

It presents new methods for 3D tissue reconstruction and cell lineage tracing applicable to diverse plant samples, overcoming previous technological limitations.

Findings

Reconstructed cellular architecture and genealogy in Calla palustris embryos and seedling shoot apex.

Revealed subcellular relations between plastids, stromules, and mitochondria in plant cells.

Identified three independent stem cell lineages in the shoot apex.

Abstract

Modern spatial microscopy has provided developmental biology with powerful research tools. However, the recent significant technological breakthroughs have inevitably led to technical "bottlenecks" for the application of the new procedures to other tasks. We have developed a protocol for the 3D reconstruction of plant organelles and tissues from stacks of two-dimensional images obtained by means of a wide range of electron transmission and light microscopy. This protocol can be applied to a large set of primary data: live, fixed or even paleobotanical material. For tissue reconstruction, we have also developed a protocol of cell lineage tracing based on the geometrical properties of plant cells. For the tissue sample consisting of $n$ cells, this approach allows the reconstruction of up to $n-1$ cell divisions from the previous cell generations. Our protocol complements the limitations of clonal analysis and recent real-time approaches of lineage tracing. It works well in the case of tissues with directed and non-directed growth. Using these protocols, the subcellular relations between the complex surface of plastids with stromules and mitochondria in the tuber parenchyma cells in \textit{Corydalis bracteata} have been revealed. Moreover, for the embryo ($n=17$) and the seedling shoot apex ($n=208$) of \textit{Calla palustris}, the cellular architecture and full cell genealogy of up to 15 cell generations have been reconstructed. Three relatively independent cell lineages with stem cells located at the surface of the shoot apex have been found. The possibility of using a wide range of software, including open-source projects, is discussed.