The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins' largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.

The proteome of the etioplast inner membranes (EPIM) of dark-grown wheat leaves (Triticum aestivum L.) was mapped as an essential part of studies on plastid differentiation. Proteins were separated by two-dimensional gel electrophoresis and analysed with mass spectrometry (MS). Over 200 protein spots were resolved and visualized by Coomassie blue staining. More than 100 spots were submitted for subsequent mass spectrometry analyses by matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) MS, electrospray tandem MS (ESI-MS/MS) or liquid chromatography-mass spectrometry (LC-MS/MS). There were 46 identified spots, from which at least 21 different proteins were identified. Among these were FtsH proteases and the peptidyl-prolyl cis-trans isomerase TLP40, as well as chloroplast coupling factor subunits and extrinsic subunits of photosystem II (PSII). Of special interest is the NADPH:protochlorophyllide oxidoreductase (POR), which is the predominant protein of prolamellar bodies, where it accumulates in a highly stable ternary complex with protochlorophyllide and NADPH. This complex is known to play an important role in the formation and dispersal of prolamellar bodies. Five different isoforms of POR, with different pI values, were identified. We discuss the possibility of these isoforms being differently phosphorylated as part of the regulation of POR-pigment complexes. The proteome mapping of EPIM is a crucial step in the understanding of the lightdependent transition of etioplasts to chloroplasts, and provides a basis for functional studies on factors influencing the greening process.