Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars

Plant phenotyping to date typically comprises morphological and physiological profiling in a high-throughput manner. A powerful method that allows for subcellular characterization of organelle stoichiometric/functional characteristics is still missing. Organelle abundance and crosstalk in cell dynamics and signaling plays an important role for understanding crop growth and stress adaptations. However, microscopy cannot be considered a high-throughput technology. The aim of the present study was to develop an approach that enables the estimation of organelle functional stoichiometry and to determine differential subcellular dynamics within and across cultivars in a high-throughput manner. A combination of subcellular non-aqueous fractionation and liquid chromatography mass spectrometry was applied to assign membrane-marker proteins to cell compartmental abundances and functions of Pisum sativum leaves. Based on specific subcellular affiliation, proteotypic marker peptides of the chloroplast, mitochondria and vacuole membranes were selected and synthesized as heavy isotope labeled standards. The rapid and unbiased Mass Western approach for accurate stoichiometry and targeted absolute protein quantification allowed for a proportional organelle abundances measure linked to their functional properties. A 3D Confocal Laser Scanning Microscopy approach was developed to evaluate the Mass Western. Two P. sativum cultivars of varying morphology and physiology were compared. The Mass Western assay enabled a cultivar specific discrimination of the chloroplast to mitochondria to vacuole relations.