RESEARCH ARTICLE A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis Rosa Pipitone1, Simona Eicke2, Barbara Pfister2, Gaetan Glauser3, Denis Falconet4, Clarisse Uwizeye4, Thibaut Pralon1, Samuel C Zeeman2, Felix Kessler1*, Emilie Demarsy1,5* 1Plant Physiology Laboratory, University of Neuchaˆtel, Neuchaˆtel, Switzerland; 2Institute of Molecular Plant Biology, Department of Biology, ETH Zurich, Zurich, Switzerland; 3Neuchaˆtel Platform of Analytical Chemistry, University of Neuchaˆtel, Neuchaˆtel, Switzerland; 4Universite´ Grenoble Alpes, CNRS, CEA, INRAE, IRIG- DBSCI-LPCV, Grenoble, France; 5Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland Abstract Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling ‘Structure Establishment Phase’ followed by a ‘Chloroplast Proliferation Phase’ during cell expansion. Moreover, these data detail thylakoid membrane expansion during *For correspondence: de-etiolation at the seedling level and the relative contribution and differential regulation of
[email protected] (FK); proteins and lipids at each developmental stage. Altogether, we establish a roadmap for
[email protected] (ED) chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.