Quantifying leaf morphogenesis in 4D

Abstract

A fundamental question in biology is how developmental processes give rise to organ shape. The wide variation in leaf shape across plant species provides an excellent model for exploring this question. Leaves can develop as a continuous leaf blade or be divided into individual leaflets. Long-standing debates exist on the contribution of different leaf regions to final leaf form and the role of tissue layers in shaping the organ. While most leaf growth studies have focused on analyzing growth at the curved organ surface, we present an advanced 3D analysis pipeline using live confocal time-lapse imaging. By applying PlantSeg’s machine learning-based 3D cell segmentation and advanced 3D image analysis in MorphoGraphX, we track individual cells over time and extract 3D growth characteristics with tissue-level annotations. We performed 3D cell segmentation of the leaf timelapse datasets from day one to seven after leaf initiation in the complex leaf of tomato (Solanum lycopersicum). By analysing growth processes in the first two layers of the leaf at the margin, we show that growth and proliferation are largely similar in the epidermis and sub-epidermis. Additionally, we examined leaflet formation in the complex tomato leaf and found that the leaflet forming cells exhibited a growth burst during leaflet emergence. We further compared development processes in the tomato leaf with another plant species that forms complex leaves - Cardamine hirsuta to identify similarities in development between species.